CN1856580A - Programmable molecular barcodes - Google Patents

Programmable molecular barcodes Download PDF

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Publication number
CN1856580A
CN1856580A CN 200480027614 CN200480027614A CN1856580A CN 1856580 A CN1856580 A CN 1856580A CN 200480027614 CN200480027614 CN 200480027614 CN 200480027614 A CN200480027614 A CN 200480027614A CN 1856580 A CN1856580 A CN 1856580A
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Prior art keywords
raman
label
method according
bar code
probe
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CN 200480027614
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Chinese (zh)
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CN1856580B (en
Inventor
X·苏
T-W·库
A·伯林
L·孙
N·桑德拉拉扬
M·山川
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英特尔公司
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Priority to US10/670,701 priority Critical
Priority to US10/670,701 priority patent/US20050064435A1/en
Application filed by 英特尔公司 filed Critical 英特尔公司
Priority to PCT/US2004/031289 priority patent/WO2005030996A2/en
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Publication of CN1856580B publication Critical patent/CN1856580B/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y10/00Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6816Hybridisation assays characterised by the detection means
    • GPHYSICS
    • G06COMPUTING; CALCULATING; COUNTING
    • G06KRECOGNITION OF DATA; PRESENTATION OF DATA; RECORD CARRIERS; HANDLING RECORD CARRIERS
    • G06K19/00Record carriers for use with machines and with at least a part designed to carry digital markings
    • G06K19/06Record carriers for use with machines and with at least a part designed to carry digital markings characterised by the kind of the digital marking, e.g. shape, nature, code
    • G06K19/06009Record carriers for use with machines and with at least a part designed to carry digital markings characterised by the kind of the digital marking, e.g. shape, nature, code with optically detectable marking
    • G06K19/06018Record carriers for use with machines and with at least a part designed to carry digital markings characterised by the kind of the digital marking, e.g. shape, nature, code with optically detectable marking one-dimensional coding
    • G06K19/06028Record carriers for use with machines and with at least a part designed to carry digital markings characterised by the kind of the digital marking, e.g. shape, nature, code with optically detectable marking one-dimensional coding using bar codes

Abstract

The present disclosure concerns methods for producing and/or using molecular barcodes. In certain embodiments of the invention, the barcodes comprise polymer backbones that may contain one or more branch structures. Tags may be attached to the backbone and/or branch structures. The barcode may also comprise a probe that can bind to a target, such as proteins, nucleic acids and other biomolecules or aggregates. Different barcodes may be distinguished by the type and location of the tags. In other embodiments, barcodes may be produced by hybridization of one or more tagged oligonucleotides to a template, comprising a container section and a probe section. The tagged oligonucleotides may be designed as modular code sections, to form different barcodes specific for different targets. In alternative embodiments, barcodes may be prepared by polymerization of monomeric units. Bound barcodes may be detected by various imaging modalities, such as, surface plasmon resonance, fluorescent or Raman spectroscopy.

Description

可设计的分子条码 Molecules can be designed barcode

发明领域 Field of the Invention

[0005] 本方法、组合物和设备涉及分子条码(molecular barcode)领域。 [0005] The present method and apparatus relates to compositions molecular barcode (molecular barcode) field. 本发明的具体实施方式涉及从有机聚合物骨架构建分子条码的方法。 DETAILED DESCRIPTION The present invention relates to a method for constructing the bar code from the organic polymer molecule backbone. 通过将标签(tag)连接于骨架上不同的位置,使用同一骨架可以产生多种分子条码。 By label (tag) attached to different positions on the backbone, the backbone may be produced using the same variety of molecules barcode. 在其它实施方式中,分子条码可以包括探针区域(probe region)和一种或多种码组分(codecomponent)。 In other embodiments, the probe molecule may comprise a barcode area (probe region) and one or more code components (codecomponent). 在其它实施方式中,分子条码可以包括聚合物拉曼标记,连接于一个或多个探针以检测靶分子。 In other embodiments, the barcode may comprise a polymer molecule Raman labels, linked to one or more probes to detect target molecules.

发明背景 BACKGROUND OF THE INVENTION

[0006] 生物分子的检测和/或鉴定可用于多种应用,用于医学诊断、法医学、毒物学、病理学、生物战、公共卫生和许多其它领域。 [0006] The detection of biological molecules and / or identification of a variety of applications, for medical diagnosis, forensics, toxicology, pathology, biological warfare, public health and numerous other fields. 虽然被研究的生物分子的主要类型是核酸和蛋白质,但对其它生物分子也感兴趣,例如糖类、脂类、多糖、脂类、脂肪酸和其它感兴趣分子。 While the main types studied biomolecules are nucleic acids and proteins, but might be interested in other biological molecules such as sugars, lipids, polysaccharides, lipids, fatty acids and other molecules of interest. 存在对快速、可靠和低成本的生物分子鉴定方法、区别相似生物分子的方法以及分析大分子复合体如病原孢子或微生物的需要。 It exists for rapid, reliable and low cost method of identifying biomolecules, biomolecule method similar distinction and macromolecular complexes to be analyzed, such as pathogenic microorganisms or spores.

[0003] 核酸检测的标准方法,例如DNA印迹法、RNA印迹法或与核酸芯片结合,都依赖于荧光、化学发光或放射探针分子与靶核酸分子的杂交。 [0003] The standard method of nucleic acid detection, for example, DNA blots, RNA blot or chip bonding to a nucleic acid, relies on a fluorescent, chemiluminescent or radioactive hybridization probe molecules and the target nucleic acid molecule. 在基于寡核苷酸杂交的测试中,在序列中与靶核酸互补的标记寡核苷酸探针被用于与核酸结合并检测核酸。 Based on the test oligonucleotide hybridization, the target nucleic acid sequence complementary to a labeled oligonucleotide probes are used in combination with a nucleic acid and detection of nucleic acids. 最近,DNA(脱氧核糖核酸)芯片已经被设计,其可以包含上百个或上千个连接的寡核苷酸探针,以结合靶核酸。 Recently, the DNA (deoxyribonucleic acid) chip has been designed, which may comprise oligonucleotide probes hundreds or thousands of linked, bind to the target nucleic acid. 不完全互补的序列之间的核酸杂交可能产生灵敏性和/或特异性问题。 Nucleic acid hybridization between complementary sequences may not fully produce sensitivity and / or specificity problems. 或者,样品中存在的低含量靶核酸可能检测不出。 Alternatively, the low levels present in the sample target nucleic acid may not be detected.

[0004] 多种技术可用于鉴定蛋白质、多肽和肽。 [0004] A variety of techniques may be used to identify proteins, polypeptides and peptides. 通常地,这些都涉及抗体的结合和检测。 Typically, these involve binding and detection antibody. 虽然基于抗体的鉴定相当快速,但这些测试有时显示高水平的假阳性或假阴性。 Although antibody-based identification is quite fast, but sometimes these tests show high levels of false positives or false negatives. 这些测试的成本高而且同时测试一个以上靶是困难的。 High cost of these tests and to test more than one target at the same time is difficult. 进一步,这些方法要求在进行测试之前,针对感兴趣的靶蛋白,制备出抗体。 Before proceeding further, these methods require during the test, for the target protein of interest, made antibodies.

[0005] 在分子生物学、基因组学、疾病诊断和药物响应预测中的许多应用都涉及核酸序列变体的鉴定。 [0005] In molecular biology, genomics, disease diagnosis, and drug response prediction Many applications involve a nucleic acid sequence variants identified. 已有的核酸测序方法,包括桑格双脱氧测序法和通过杂交测序,都趋于相对缓慢、昂贵、费力而且可能要用到放射标签或其它有毒化学品。 Conventional nucleic acid sequencing methods, including sequencing and Sanger dideoxy sequencing by hybridization, have tended to be relatively slow, expensive, laborious and may use a radiation label or other toxic chemicals. 已有的方法也受限于在一个反应中所获得的序列信息数量,通常到大约1000个碱基或更少。 Conventional methods are also limited to the number in a sequence information obtained in the reaction, usually to about 1000 bases or less. 存在对更加快速、成本有效的和自动化的核酸测序方法的需要。 There is a need for more rapid, cost-effective and automated nucleic acid sequencing methods.

附图简述 BRIEF DESCRIPTION

[0006] 以下的附图形成本说明书的一部分,并且被包括以进一步示范说明本发明公开实施方式的某些方面。 [0006] The following drawings form part of the present specification and are included to further illustrate exemplary embodiments of certain aspects of the disclosed embodiment of the present invention. 通过参照一个或多个这些附图,并结合这里提供的详细说明,可以较好地理解这些实施方式。 By reference to one or more of these drawings, and the detailed description provided herein, it will be better understood embodiments.

[0007] 图1说明用支链120和标签130修饰的有机骨架110产生条码100的示例性方法。 [0007] Figure 1 illustrates a tag 130 and 120 branched chain organic backbone modified exemplary method 110 generates the barcode 100. 条码100可以包括能与靶结合的探针部分150。 Barcode section 100 may include a probe capable of binding to the target 150. 标签130可以接受另外的修饰,例如通过与抗体140结合。 Tag 130 may receive an additional modification, for example by binding to the antibody 140.

[0008] 图2说明用同一骨架产生不同条码201、202、203的示例性方法。 [0008] Figure 2 illustrates an exemplary method different barcodes 201, 202 generating the same skeleton. 标签240、250、260可以被放在不同位置,以产生可区分的条码201、202、203。 Tags can be placed in different positions 240,250,260, 201, 202 to generate a barcode distinguishable. 条码201、202、203与靶的结合可以通过连接于条码201、202、203上的探针部分210、220、230进行介导。 Barcode 202, 203 may be performed by binding the target probe portion is connected to a bar code 201, 210, 220 mediate.

[0009] 图3说明具有单链核酸骨架的几个条码301、302、303、304的例子。 [0009] FIG. 3 illustrates an example of a single-stranded nucleic acid backbone 301, 302 of several symbols. 标签310、320、330在骨架上各个不同位置被添加,以产生不同的光谱,为例如拉曼光谱所鉴定。 Tags 320, 330 are added at various locations on the skeleton, to produce different spectra, Raman spectra, for example, identified. 在条码302、303、304上不同位置连接同一标签330的条码可产生可区别的拉曼光谱。 It may generate distinguishable Raman spectra at different locations connected to the same bar code on the bar code label 330 to 302, 303.

[0010] 图4说明图3中公开的条码所产生的拉曼光谱的例子。 [0010] Examples of bar codes disclosed in FIG. 3 FIG. 4 illustrates a Raman spectrum generated. 条码301、302、303和304出现在同一图中。 Bar codes 302, 303 and 304 appear on the same graph.

[0011] 图5说明用连接有一个或多个标签510的已知序列的多个短寡核苷酸520产生条码的示例性方法。 [0011] Figure 5 illustrates an exemplary method of connecting with a bar code 520 to produce one or more of a plurality of short oligonucleotides of known sequence tags 510 nucleotides. 通过与模板分子500杂交,可将寡核苷酸-标签分子组合成条码。 Template molecules by hybridization 500, may be an oligonucleotide - Barcode tag molecule composition. 模板500可包括用于寡核苷酸-标签杂交的容器部分(containersection)540和用于与靶分子如核酸结合的探针部分550。 Template 500 may include an oligonucleotide - hybridized tag container section (containersection) 540 and a nucleic acid target molecule binding probe portion 550. 在可选的实施方式中,探针550可包括,例如,可以与蛋白质、肽或其它靶类型结合的适体序列。 In an alternative embodiment, the probe 550 may include, for example, may be combined with a protein, peptide or other target types aptamer sequences.

[0012] 图6表示产生条码的示例性方法的示意图,包括:通过将标签部分连接于寡核苷酸或核酸,构建码组分601、602、603、604;形成模板606以及将码组分与模板605杂交,形成条码607。 ; Forming a template 606 and the code components by the label moiety is attached to an oligonucleotide or nucleic acid construct components 601,602,603,604 code: [0012] FIG. 6 is a diagram of an exemplary method of generating barcodes, comprising represents 605 hybridize with the template to form a bar code 607.

[0013] 图7表示用图6的方法所产生的条码鉴定互补靶链(complementarytarget strand)存在与否的示例性方法的示意图。 [0013] FIG. 7 shows a schematic diagram of an exemplary method of the presence or absence of a bar code identifying complementary target strand (complementarytarget strand) using the method of FIG. 6 produced.

[0014] 图8表示几个拉曼标签801、802、803、804、805、806产生的SERS(surface enhanced Raman spectroscopy(表面增强拉曼光谱))光谱图的例子。 [0014] FIG. 8 shows an example of SERS (surface enhanced Raman spectroscopy (SERS)) Several Raman spectra generated label 801,802,803,804,805,806.

[0015] 图9说明聚合物拉曼标记910的例子。 [0015] Figure 9 illustrates an example of polymeric Raman label 910. 单体单元901、902通过共价键906相连,该共价键由连接于骨架909的功能基904、908与生长聚合链的末端处另一功能基904、908的相互作用产生。 Monomeric units 901, 902, 906 are connected by a covalent bond, the covalent interactions generated by another functional group attached to the functional group at the end of skeleton 904, 908, 909 and 904, 908 of the chain growth polymerization. 任选地,可添加另外的单元903。 Optionally, add an additional 903 units.

[0016] 图10表示产生聚合物拉曼标记的示例性方法的示意图。 [0016] FIG. 10 shows a schematic diagram of an exemplary method of generating Raman label polymer. 用固体支持物1001附着组分1005(例如聚合物拉曼标记的一部分)。 1001 is attached with a solid support component 1005 (e.g., polymeric Raman label part). 组分1005的开口端1104被去保护,并通过单体单元1010的去保护功能基1006,将单体单元1010与组分1005连接。 1005 1104 open end of the component is deprotected and the deprotected functional group of monomer units 1006 through 1010, the components 1005 and 1010 monomeric units connected. 拉曼标签1002、1003、1008被连接于聚合物拉曼标记上。 Raman tag 1002,1003,1008 Raman labels are attached to the polymer.

[0017] 图11A表示产生聚合物拉曼标记1105的另一示例性方法。 [0017] FIG. 11A shows another exemplary method 1105 of generating Raman label polymer. 第一反应用于将功能基1102a、1102b连接于拉曼标签1101a、1101b,产生功能化拉曼标签1103a、1103b。 A first functional group for reaction 1102a, 1102b is connected to a Raman label 1101a, 1101b, to create functional Raman labels 1103a, 1103b. 第二反应用于聚合功能化拉曼标签1103a、1103b,以形成次级聚合物拉曼标记1104a、1104b。 The second reaction is used for the polymerization of functional Raman labels 1103a, 1103b, to form the secondary polymer Raman labels 1104a, 1104b. 每个次级聚合物拉曼标记1104a、1104b包括预先确定数目的单体拉曼标签1103a、1103b。 Each secondary polymer Raman labels 1104a, 1104b monomers comprising a predetermined number of Raman labels 1103a, 1103b. 在本实施例中,第一次级聚合物1104a包括第一单体1103a的“n”个拷贝,而第二次级聚合物1104b包括第二单体1103b的“m”个拷贝。 In the present embodiment, the first polymer secondary 1104a comprises "n" copies of the first monomer 1103a, 1104b and the second polymer comprises a secondary "m" copies 1103b of the second monomer. 将预先确定比例的次级聚合物拉曼标记1104a、1104b混合并交联,形成聚合物拉曼标记1105。 The predetermined percentage of the secondary polymer Raman labels 1104a, 1104b are mixed and crosslinked to form a polymeric Raman label 1105.

[0018] 图11B表示产生聚合物拉曼标记的又一示例性方法。 [0018] FIG 11B shows yet another exemplary method of generating Raman label polymer. 具有功能基1112的聚合物分子1109与不同的拉曼标签1110结合,形成聚合物拉曼标记1111。 Polymer molecules having a functional group 1109 1112 1110 combined with different Raman tags, Raman label 1111 to form a polymer. 每个类型的拉曼标签1110的数目被预先确定,以产生具有特异化光谱性质的聚合物拉曼标记1111。 Raman tag number of each type 1110 is determined in advance, to produce a polymer with a specific Raman label 1111 of spectral properties.

[0019] 图12说明与一个或多个探针1206相连以鉴定靶分子的聚合物拉曼标记的几个例子。 [0019] Figure 12 illustrates a few examples of the polymer Raman identify target molecules labeled with one or more probes 1206 are connected. 第一个例子1201显示通过接头1205与探针1206连接的聚合物拉曼标记1204。 The first example display 1201 connected by a linker polymers Raman probe 1206 1205 1204 labeled. 第二个例子1202显示两个聚合物拉曼标记1204,其通过1205与纳米颗粒1207相连,而另外的接头1205将纳米颗粒1207与两个探针1206连接。 The second example shows two polymeric Raman label 1202 1204, which is connected with the nanoparticle 1207 through 1205, and the other nanoparticle linker 1205 1207 1206 connected to the two probes. 第三个例子1203显示通过接头1205与纳米颗粒连接的多个探针1206,并且多个拉曼标签1208与纳米颗粒1207连接。 A third example of displaying a plurality of probes 1203 and 1206 are connected via a linker nanoparticles 1205, 1208 and connected to the plurality of Raman tags 1207 nanoparticles.

[0020] 图13表示修饰的核酸,腺嘌呤的几个拉曼标签产生的SERS(表面增强拉曼光谱)光谱图的例子。 [0020] FIG. 13 shows a modified nucleic acid, several examples of Raman SERS label producing adenine (surface enhanced Raman spectroscopy) spectrum of FIG.

说明性实施方式的描述 Description of illustrative embodiments

[0021] 下面详细的描述包含许多具体的细节,以提供对本发明公开实施方式的更透彻的理解。 [0021] The following detailed description contains numerous specific details in order to provide a more thorough understanding of the disclosed embodiments of the present invention. 然而,对本领域所属普通技术人员来说,显而易见的是,这些实施方式可以在没有这些具体细节的情况下进行实施。 However, those of ordinary skill in this art, it will be apparent that these embodiments can be practiced without these specific details. 在其它的情况下,本领域所熟知的设备、方法、过程和各个组件在这里没有被详细描述。 In other instances, well known in the art devices, methods, procedures, and individual components have not been described in detail herein.

定义 definition

[0022] 如这里所使用,“一个(a)”或“一个(an)”可以意味着某项目的一个或多个。 [0022] As used herein, "a (A)" or "a (AN)" may mean one or more of an item.

[0023] 如这里所使用,项目的“多种(multiplicity)”意味着两个或多个项目。 [0023] As used herein, the item "variety (the multiplicity)" means two or more items.

[0024] 如这里所使用,“核酸”包括DNA、RNA(核糖核酸),可为单链的、双链的或三链的及其任何化学修饰。 [0024] As used herein, "nucleic acid" encompasses DNA, RNA (ribonucleic acid), may be single-stranded, double-stranded or triple stranded and any chemical modifications. 事实上,核酸的任何修饰可以被考虑在内。 In fact, any modification of the nucleic acid can be taken into account. “核酸”可以具有几乎任何长度,从2个或更多个碱基的寡核苷酸到全长度的染色体DNA分子。 "Nucleic acid" may have almost any length, from oligonucleotides of nucleotides 2 or more bases into the chromosomal DNA of the full length molecule. 核酸包括但不限于寡核苷酸和多核苷酸。 Nucleic acids include but are not limited to oligonucleotides and polynucleotides.

[0025] “探针”分子是表现出与一个或多个靶选择性和/或特异性结合的任何分子。 [0025] "probe" molecule is any molecule that exhibits binding with one or more target selectivity and / or specificity. 在本发明的各种实施方式中,将各个不同的探针分子与可区分条码连接,以便检测与来自一组不同探针的一个具体探针的结合作用。 In various embodiments of the present invention, the various probe molecule attached to the bar code can be distinguished, in order to detect a specific binding with the probe from a set of different probes. 这些实施方式对于所用探针分子的类型是没有限制的。 For these embodiments, the type of probe molecule is not limited. 可以使用本领域所知的任何探针分子,包括但不限于寡核苷酸、核酸、抗体、抗体片段、结合蛋白、受体蛋白、肽类、凝集素、底物、抑制物、激活子、配体、激素、细胞因子等。 Any probe molecules known in the art may be used, including but not limited to oligonucleotides, nucleic acids, antibodies, antibody fragments, binding proteins, receptor proteins, peptides, lectins, substrates, inhibitors, activators, ligands, hormones, cytokines, and the like. 在某些实施方式中,探针可包括已共价地或非共价地与一个或多个条码连接以鉴定不同靶的抗体、适体、寡核苷酸和/或核酸。 In certain embodiments, the probe may comprise been covalently or non-covalently linked to one or more different target barcode to identify antibodies and aptamers, oligonucleotides and / or nucleic acids.

说明性实施方式 Illustrative Embodiment

[0026] 所公开的方法、组合物和设备用于生物分子如核酸和蛋白质的检测、鉴定和/或加标签。 [0026] The disclosed methods, compositions and devices for biomolecules such detection, identification and / or labeling nucleic acids and proteins. 在本发明的具体实施方式中,可使用这些方法、组合物和设备,通过对骨架进行各种修饰,由单个有机骨架(single organic backbone)产生多个条码。 In a specific embodiment of the present invention may be used such methods, compositions and devices, through various modifications of the skeleton, produced by a single bar code a plurality of organic framework (single organic backbone). 这些实施方式并不限于单个骨架,而可以使用一个或多个不同的骨架。 These embodiments are not limited to a single frame, and may use one or more different backbone. 优势包括:通过改变标签沿骨架的连接位置,用同一骨架产生不同的条码的能力。 Advantages comprising: changing the connection position of the label along the backbone, the ability of generating different barcode same skeleton. 其它实施方式涉及产生聚合物拉曼标记,以快速鉴定生物分子或加标签于生物分子。 Other embodiments relate to produce a polymer Raman labels to quickly identify tagged biomolecule or biomolecules. 其它优势包括多肽的灵敏性和精确性检测和/或鉴定。 Other advantages include the sensitivity and accuracy of detection of the polypeptide and / or identification.

条码合成(Barcodes by Synthesis) Barcode synthesis (Barcodes by Synthesis)

[0027] 在本发明的一个实施方式中,如图1所示,条码骨架(barcodebackbone)110可由包括有机结构的聚合物链形成,该聚合物链包括核酸、肽、多糖和/或化学衍生聚合物序列的任何组合。 [0027] In one embodiment of the present invention, shown in Figure 1, the bar code bobbin (barcodebackbone) 110 may include an organic polymer chain structure is formed, the polymer chain comprises a nucleic acid, peptide, polysaccharide, and / or chemically derivatized polymeric the composition of any sequences. 在某些实施方式中,骨架110可包括单链或双链核酸。 In certain embodiments, the bobbin 110 may comprise single or double stranded nucleic acid. 在一些实施方式中,骨架可与探针部分150,例如寡核苷酸、抗体或适体连接。 In some embodiments, the backbone may be, for example, oligonucleotide, antibody or aptamer portion 150 is connected to the probe. 骨架110可用一个或多个支链结构(branch structure)120进行修饰,形成额外的形态多样性和标签附着位置。 Skeleton 110 using one or more branched structure (branch structure) 120 modified, to form additional morphological diversity and label attachment position. 支链结构120可用本领域熟知的技术形成。 Branched structure 120 may be formed by the technique known in the art. 例如,在条码100包括双链核酸的情况下,支链结构120可通过寡核苷酸的合成并与单链模板核酸的杂交而形成。 For example, in the case 100 includes a bar code double stranded nucleic acid, a branched structure 120 may be of synthetic oligonucleotides and hybridized with a single-stranded template nucleic acid is formed. 寡核苷酸可这样设计,使得序列的一部分(例如5′末端)与模板互补而另一部分(例如3′末端)不与模板互补。 Oligonucleotides may be designed such that the portion of the sequence (e.g. the 5 'end) and the other portion is complementary to the template (e.g., 3' end) is not complementary to the template. 因此,条码100将包含双链序列的片段和单链支链结构120的短片段。 Thus, the bar code 100 containing short segments of double-stranded fragments and single-stranded sequences branched structure 120. 如图1所公开,例如通过标记130的寡核苷酸的杂交,可将标签130添加到条码上,该寡核苷酸与支链结构120的单链部分在序列上是互补的。 FIG 1 discloses, for example by oligonucleotide hybridization markers 130, 130 may be added to the bar code label, single-stranded portion of the oligonucleotide is a branched structure 120 is complementary in sequence.

[0028] 可用寡核苷酸模拟产生有机骨架(organic backbone)110。 [0028] generating an analog oligonucleotides can be used organic framework (organic backbone) 110. 可用新的基团取代糖类和核苷酸单元的核苷间键合(internucleoside linkage)即骨架。 Available new substituted sugars and internucleoside bond nucleotide units (internucleoside linkage) i.e. skeleton. 探针150可被用于与合适的核酸靶化合物杂交。 Suitable probes 150 may be used to hybridize with the nucleic acid target compound. 已显示具有优异杂交性质的寡聚化合物或寡核苷酸模拟的一个例子被称为肽核酸(peptide nucleic acid(PNA))。 An example has been shown to have excellent hybridization properties of an oligomeric compound or an oligonucleotide analog is referred to as a peptide nucleic acid (peptide nucleic acid (PNA)). 在PNA化合物中,寡核苷酸的糖骨架被含酰胺的骨架取代,例如氨乙基甘氨酸骨架。 In PNA compounds, the sugar-backbone of an oligonucleotide is substituted with an amide containing backbone, for example an aminoethylglycine backbone. 在此例子中,核碱基(nucleobase)被保留并直接或间接地与骨架酰胺部分的氮杂氮原子相连。 In this example, the nucleobase (the nucleobase) is retained and is connected directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. 几件美国专利公开了PNA化合物的制备过程,包括例如US 5,539,082;US 5,714,331和US5,719,262。 Several U.S. patents disclose the preparation of PNA compounds include, for example US 5,539,082; US 5,714,331 and US5,719,262. 除此,PNA化合物也在Nielsen等(Science,1991,254,1497-15)中被公开。 In addition, PNA compounds are also Nielsen et al (Science, 1991,254,1497-15) is disclosed in.

[0029] 为了将一个条码100与另一个区分开来,可将标签130直接加到骨架110上,或者直接添加到一个或多个支链结构120上。 [0029] To a bar code 100 to separate from another, the label 130 can be directly applied to the bobbin 110, or added directly to one or more of a branched structure 120. 条码100可通过将另一分子140(例如抗体)与一个或多个标签130连接而被进一步修饰。 Barcode 100 may be connected to one or more tags 130 are further modified by another molecule 140 (e.g., antibody). 在使用大体积基团的情况下,连接于支链位置120的标签部分130的修饰会为与靶分子相互作用的探针150提供较低的空间位阻。 In the case where a bulky group connected to the branch location label 120 will provide a lower steric hindrance to interact with a target molecule 150 modified probe portion 130. 标签130可通过成象方法,例如荧光显微法、FTIR(傅里叶变换红外)光谱、拉曼光谱、电子显微法以及表面等离子体共振法(surface plasmon resonance)被读出。 Tag 130 by image forming methods, such as fluorescence microscopy, FTIR (Fourier Transform Infrared) spectroscopy, Raman spectroscopy, electron microscopy and surface plasmon resonance (surface plasmon resonance) is read out. 已知各种不同的成象可检测标签130的形态学、拓扑学、化学和/或电学性质,包括但不限于传导性、隧穿电流、电容电流等。 Various known detectable morphological imaging, topology, chemical and / or electrical properties of tags 130, including but not limited to conductivity, tunneling current, capacitive current, etc. 所用的成象方法取决于标签部分130的性质以及产生的最终信号。 The image forming method used depends on the nature of the tag portion 130 and the final signal generated. 不同类型的已知标签130,包括但不限于荧光、拉曼、纳米颗粒、纳米管、富勒烯和量子点标签130,通过其拓扑学、化学、光学和/或电子性质,可用于鉴定条码100。 Different types of known tags 130, including but not limited to fluorescent, Raman, nanoparticle, nanotubes, fullerenes and quantum dot tags 130, through which the topology, chemical, optical and / or electronic properties, can be used to identify the barcode 100. 这些性质既作为所用标签部分130的类型,又作为标签130在骨架110或支链结构120上的相对位置的函数而发生变化,导致为每个条码100产生可区分的信号。 These properties are used both as tag type portion 130, the tag 130 varies in function or the relative position of the bobbin 110 on the structure 120 and as a chain, resulting in the bar code 100 is generated for each distinguishable signals.

[0030] 如图2所示,识别特定靶的不同探针210、220、230可被连接于可区分条码201、202、203。 [0030] As shown in FIG 2, to identify a particular target different probes 210, 220 may be connected to a bar code 201, may be distinguished. 在此示例性实施方式中,多个标签240、250、260在不同位置被连接于条码201、202、203。 In this exemplary embodiment, the plurality of tags 201, 202, 240,250,260 are connected to a bar code in a different location. 标签240、250、260可包括,例如,拉曼标签或荧光标签。 240,250,260 tag may comprise, for example, a Raman label or a fluorescent label. 由于相邻标签可能相互作用,例如通过荧光共振能量转移(FRET)或其它机理,因此从同一套标签部分240、250、260获得的信号可能依赖于标签240、250、260之间的位置和距离而发生变化(参见实施例1)。 Since the adjacent label may interact, for example, transfer (FRET) or fluorescence resonance energy through other mechanisms, therefore may depend on the position and the distance between the tag signal from the same set of labels 240,250,260 240,250,260 portion obtained vary (see Example 1). 所以,具有相似或同一骨架的条码201、202、203可被可区分地标记。 Therefore, the bar code having the same skeleton 201, similar or may be distinguishably labeled. 通过将探针210、220、230,例如抗体、适体或寡核苷酸,连接于条码201、202、203,可提供结合靶分子的特异性。 By the probe 210, 220, such as antibodies, aptamers or oligonucleotide, 201, 202 is connected to the bar code may be provided that specifically binds target molecule. 由于对应于给定探针210、220、230特异性的条码201、202、203信号是已知的,因此可能通过测定哪个探针210、220、230与样品中靶相结合而分析分子复合混合物并检测各个种类。 Since the probes 210, 220 corresponding to a specific bar code 201, the signal is known, it may be combined with the target sample 210, 220, by which the measuring probe molecule complex mixture analysis and detecting the respective kinds.

[0031] 在本发明的某些实施方式中,如图1和图2所示,条码100、201、202、203的骨架110由磷酸二酯键、肽键和/或糖苷键组成。 [0031] In certain embodiments of the present invention, FIGS. 1 and 2, the bar code 110 by the skeleton 100,201,202,203 phosphodiester bonds, peptide bonds and / or glycosidic linkages. 例如,可使用标准的亚磷酰胺化学,形成包括DNA链的骨架110。 For example, using standard phosphoramidite chemistry, form the skeleton 110 includes a DNA strand. 形成由磷酸二酯连接的骨架110的其它方法是已知的,例如聚合酶链反应(PCRTM)扩增。 Other methods of connection are formed by a phosphodiester backbone 110 are known, such as polymerase chain reaction (PCRTM) amplification. 骨架110的末端可具有不同的功能基,例如生物素、氨基、醛基或硫羟基。 End of the bobbin 110 may have different functional groups, such as biotin, amino, aldehyde or thiol. 这些功能基可用于连接探针部分150、210、220、230,或者用于附着标签130、240、250、260。 These functional groups can be used to connect the probe portion 150,210,220,230, 130,240,250,260 or for attaching labels. 标签130、240、250、260可被进一步修饰,以获得不同的尺寸、电学或化学性质,便于检测。 Tag 130,240,250,260 may be further modified to obtain different sizes, electrical or chemical properties to facilitate detection. 例如,可使用抗体,与地高辛或荧光素标签130、240、250、260结合。 For example, an antibody may be used, 130,240,250,260 combination with fluorescein or digoxigenin label. 可使用链霉抗生物素与生物素标签130、240、250、260结合。 Using streptavidin biotin binding tag 130,240,250,260. 可将金属原子沉积在条码100、201、202、203结构上,例如通过使用酶标签130、240、250、260催化还原金属离子溶液来实现。 Metal atoms may be deposited on the bar code 100,201,202,203 structure, for example by using an enzyme label 130,240,250,260 catalytic reduction of metal ions in solution. 在条码100、201、202、203包括肽部分的情况下,可对肽进行磷酸化,以形成标签130、240、250、260修饰140。 In the case 100,201,202,203 bar code portion comprises a peptide, a peptide may be phosphorylated to form a label modified 130,240,250,260 140. 修饰的140标签130、240、250、260可被多种本领域已知的技术检测。 130,240,250,260 modified tag 140 may be a variety of detection techniques known in the art.

[0032] 在本发明的某些实施方式中,为安全跟踪(security tracking)之目的,可将包含一个或多个条码100、201、202、203的溶液应用于对象。 [0032] In certain embodiments of the invention, the security track (security tracking) purposes, may comprise a solution of one or more barcode 100,201,202,203 applied to the object. 这些方法在本领域是已知的。 These methods are known in the art. 例如,一个英国公司(SmartWater Ltd.)已经开发出用包含数字DNA(digital DNA)链的流体标记贵重物品的方法。 For example, a UK company (SmartWater Ltd.) methods have been developed using DNA comprising a digital fluid (Digital DNA) labeled strand valuables. 该DNA的确不可能从物品上冲洗掉,而且可用于独特地鉴定昂贵物品或祖传遗物。 The DNA does not possible rinsed from the article, but also can be used to uniquely identify an article or heirlooms expensive. 该DNA可以被任何法医实验室检测。 The DNA can be any forensic laboratory. 这些方法也可用于标记带有如此处所公开的分子条码100、201、202、203的项目。 These methods can also be used to label the molecule with a barcode item of 100,201,202,203 disclosed herein. 在这些应用中,检测条码100、201、202、203不需要基于DNA序列的法医分析。 In these applications, the bar code is detected based 100,201,202,203 forensic analysis of DNA sequences required.

条码杂交 Barcode Hybrid

[0033] 在本发明的其它实施方式中,如图5所示,涉及通过杂交产生条码530的方法。 [0033] In other embodiments of the present invention, shown in Figure 5, it relates to a method of producing the bar code 530 by hybridization. 在此实施方式中,条码530包括与寡核苷酸520杂交的核酸500。 In this embodiment, the bar code 530 520 comprising a nucleic acid with an oligonucleotide 500 hybridizes. 一个或多个标签部分510可被连接于已知序列的寡核苷酸520,该已知序列可例如通过已知的化学合成技术而产生。 One or more tag portion 510 may be connected to a known oligonucleotide sequence 520, for example, the known sequence can be produced by known chemical synthesis techniques. 产生加标签的寡核苷酸520的各种方法在本领域是熟知的。 Generating an oligonucleotide tagged nucleotide 520 of the various methods are well known in the art. 通过一系列加标签的寡核苷酸520与单链DNA模板500的杂交,形成条码530。 Through a series of oligonucleotide hybridization with single-stranded DNA template 520 tagged 500, 530 form a bar code. 模板500包括容器部分540和探针部分550。 Template 500 includes a container portion 540 and probe portion 550. 探针部分550被设计与互补靶核酸序列杂交。 Probe portion 550 is designed to hybridize to a complementary target nucleic acid sequence. 可选地,探针部分550可包括可以与蛋白质、肽或其它靶生物分子结合的适体序列。 Alternatively, portion 550 may include a probe aptamer sequences may be combined with a protein, peptide or other target biomolecule. 在各种实施方式中,探针区域540可以有2到30、4到20或14到15个核苷酸长。 In various embodiments, the probe region 540 may have 2 to 30,4 to 20 or 14 to 15 nucleotides long. 探针550长度没有限制,2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、125、150、200、250个核苷酸或甚至更长的探针部分550被考虑在内。 The length of the probe 550 is not limited, 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35 , 40,45,50,55,60,65,70,75,80,85,90,95,100,125,150,200,250 nucleotides or even longer portion of the probe 550 is considered Inside.

[0034] 图3说明用于本发明各种实施方式的示例性拉曼标记寡核苷酸。 [0034] FIG. 3 illustrates an embodiment of the present invention, various embodiments of an exemplary Raman labeled oligonucleotide. 拉曼标签310、320、330被连接于同一寡核苷酸序列的不同核苷酸,以产生不同的光谱(图4)。 Raman labels 320, 330 are connected to the same oligonucleotide sequence different nucleotide to produce a different spectrum (FIG. 4). 例如,寡核苷酸302、303和304表示同一寡核苷酸序列,其中标签330的位置有所改变。 For example, the oligonucleotides 302, 303 and 304 represent the same oligonucleotide sequence, wherein the change of position of the tag 330. 如图4所示,图3公开的标记寡核苷酸301、302、303、304的拉曼光谱是可以区别的。 4, FIG. 3 labeled oligonucleotides disclosed Raman spectra 301, 302 are distinguishable. 图4表明,连接在同一寡核苷酸序列302、303、304上的同一拉曼标签330的位置的微小变动可以产生不同类型的拉曼光谱(更详细地,参见下面的实施例1和2)。 Figure 4 shows that small changes connected to the same oligonucleotide sequence 302, 303 in the same position of the Raman tag 330 may generate different types of Raman spectroscopy (in more detail, with reference to the following Examples 1 and 2 ).

[0035] 在图5所示的本发明实施方式中,当将一个或多个标记寡核苷酸520与模板分子500的容器部分540杂交时,形成了条码530。 [0035] The embodiment of the invention shown in FIG. 5, when one or more labeled oligonucleotides hybridize to portions 540 and 520 of the container 500 of the template molecule, a barcode 530 is formed. 标记寡核苷酸520的序列被设计与容器部分550成互补,而不与探针部分550为互补。 520 labeled oligonucleotide sequence is designed to be complementary with the container portion 550, portion 550 is not complementary to the probe. 通过杂交与模板500结合的标签部分510的组合被选择用于提供可区分的信号。 Label portion 510 by a combination of hybridization and binding of the template 500 is selected for providing a signal distinguishable. 对可以使用的信号类型没有限制,而且可以使用任何已知的检测技术,包括但不限于拉曼光谱、FTIR、表面等离子体共振。 No limitation on the type of signal that can be used, and any known detection technique, including but not limited to Raman spectroscopy, FTIR, surface plasmon resonance. 杂交之后,通过已知的方法,包括但不限于超滤法、HPLC(高效液相色谱)、羟基磷灰石柱色谱法、超离心法等,将条码530从未杂交的寡核苷酸520和模板链500中分离。 After hybridization, by known methods, including but not limited to ultrafiltration, HPLC (High Performance Liquid Chromatography), hydroxyapatite column chromatography, ultracentrifugation, etc., the bar code 530 will never hybridized oligonucleotides 520 separation and 500 in the template strand. 产生条码530的这种方法与标准技术比较,具有高的标记效率并且需要减少数量的标记寡核苷酸520,其中每个标记寡核苷酸520包括分离且可鉴别的条码530。 This method generates the barcode 530 is relatively standard techniques, having a high labeling efficiency and the need to reduce the number of labeled oligonucleotides 520, 520 wherein each labeled oligonucleotide includes a separator 530 and may identify the barcode. 正如对技术人员显而易见的是,图5所示的方法表明了产生条码530的组合方法,允许采用较小数量的标记寡核苷酸520,形成大量的可区分条码530。 As is apparent to the skilled person, the method shown in FIG. 5 shows a method of producing a combination of barcode 530, allowing the use of a smaller number of labeled oligonucleotides 520, forming a large number of bar code 530 can be distinguished.

[0036] 在图5所示的本发明某些实施方式中,模板500序列的长度可以从探针部分550以及与容器部分540杂交的标记寡核苷酸520的大小进行测定。 [0036] In certain embodiments of the present invention shown in FIG. 5, the length of the sequence template 500 may be the size of 520 nucleotides from the probe portion 550 and a portion of the container 540 labeled oligonucleotide hybridization is measured. 例如,对于长度为“n”个碱基的探针部分550和长度为“m”个碱基的各个标记寡核苷酸520而言,模板500的长度等于(1+m)乘以n(或可选地,(n乘以m)+n)。 For example, the length of "n" and the length of the probe portion 550 bases of the "m" of each labeled oligonucleotide base 520, the length of template 500 is equal to (1 + m) is multiplied by n ( or alternatively, (n multiplied by m) + n). 例如,假设探针部分550的长度为9个碱基,标记寡核苷酸520的长度为5个碱基,则用于给所有可能的9-mer(9聚体)探针序列提供独特条码的所需模板500长度为(1+5)乘以9个,或54个碱基。 For example, assuming the length of the probe portion 550 is 9 bases, labeled oligonucleotide length is 5 bases 520, it is used to provide a unique bar code to all possible 9-mer probe sequences (9-mer) the template 500 of the desired length (5 + 1) multiplied by 9, or 54 bases.

[0037] 如果允许部分序列重叠,则给定的54碱基模板可包含最多达50个不同的5-mer(5聚体)序列,条件是全杂交(即,5-mer不能只与模板500的最后4个碱基结合)。 [0037] If a partial sequence overlap, a given base in the template 54 may comprise up to fifty different 5-mer (5-mer) sequence, with the proviso that the full hybridization (i.e., 5-mer template 500 allows not only to binding last 4 bases). 包含在这样的模板中的可能的不同m-mer(m聚体)数目也可被计算为等于(n+(n乘以m)-m+1)。 Possible different m-mer (m mer) contained in such a template may be calculated as the number equal to (n + (n multiplied by m) -m + 1). 另一方面,存在45(或1024)个可能的5-mer(5聚体)序列可以被合成,这是由于5-mer的每个位置可包含4个可能的碱基之一,而且有5个位置。 On the other hand, the presence of 45 (or 1024) possible 5-mer (5-mer) sequence may be synthesized, which is due to the 5-mer each position may comprise one of the four possible bases, and 5 positions. 这意味着,有4m-(n+(n乘以m)-m+1)种5-mer可以被用作码组分。 This means that it 4m- (n + (n multiplied by m) -m + 1) 5-mer species can be used as component codes. 在目前情况下,有974(1024-50)种5-mer可作为码组分。 In the present case, there are 974 (1024-50) 5-mer species can be used as component codes. 容器部分540将被设计与来自974个有效类型的系列独特5-mer杂交。 The container portion 540 will be designed with a unique series of 5-mer hybridized from 974 valid type. 将包括合适条码序列的标记寡核苷酸520引入并与容器部分540杂交。 The labeled oligonucleotide comprising a nucleotide sequence of a suitable bar code portion 520 and introduced into the container 540 hybridized. 每个标记的寡核苷酸520将包含提供独特信号的标签,以便可以与其它码组分区别开来。 Each labeled oligonucleotide 520 will provide a unique signal comprises a label, so that the component can start to distinguish other codes.

[0038] 通过参见示例性的说明,可以阐明原理。 [0038] Referring to example by way of illustration, it is possible to clarify the principle. 如果探针部分550是4个碱基长(n=4),标记寡核苷酸520包括3个碱基序列(m=3),则模板500长度为16个碱基长((1+3)乘以4)。 If the probe portion 550 is 4 bases in length (n = 4), labeled oligonucleotides 520 includes three base sequence (m = 3), the template 500 a length of 16 bases in length ((1 + 3 ) multiplied by 4). 这导致12个碱基的容器部分540和4个碱基(4-mer)的探针部分550。 This causes the container 540 portion of 12 bases and 4 bases (4-mer) probe portion 550. 由于m=3,所以有64(43)个可能的有效3-mer(3聚体)序列。 Since m = 3, so there are 64 (43) effective possible 3-mer (3-mer) sequence. 各个16碱基模板500可包含达14个3-mer类型(4+(3*4)-3+1=14)。 16 each base 500 may comprise a template 14 of the type 3-mer (4 + (3 * 4) + 1 = 14 -3). 任意的模板500序列示于下面的SEQ ID NO:1中,其中探针部分550(下划线)在左,容器部分540在右。 Any template sequence 500 is shown below in SEQ ID NO: 1, wherein the probe portion 550 (underlined) in the left part of the container 540 on the right.

AGAAAGT ACA TAT GTC(SEQ ID NO:1) AGAAAGT ACA TAT GTC (SEQ ID NO: 1)

[0039] 在此例子中,16-mer(16聚体)包含14个不同的3-mer(3聚体)序列(AGA GAA AAA AAG AGT GTA TAC ACA CAT ATA TAT ATG TGT GTC),这是因为3-mer中没有相同的。 [0039] In this example, 16-mer (16-mer) containing 14 different 3-mer (3-mer) sequence (AGA GAA AAA AAG AGT GTA TAC ACA CAT ATA TAT ATG TGT GTC), because 3-mer is not the same. 为防止码组分在错误的位置结合,需要至少18个不同类型(=14+4)独特标记的3-mer条码序列,以可区分地标记所有可能的4-mer探针序列550。 Code to prevent binding components in the wrong position, at least 18 different types (= 14 + 4) 3-mer sequences unique bar code labeled distinguishably labeled to all possible 4-mer probe sequence 550. (所需独特码组分的数目可被计算为等于((2乘以n)+(n乘以m)-m+1))。 (The number of unique codes necessary components may be calculated to be equal to ((2 multiplied by n) + (n multiplied by m) -m + 1)). 对于SEQ ID NO:1所公开的具体容器序列540,只需要4个标记的3-mer-TCA、TGT、ATG和CAG。 For SEQ ID NO: 1 disclosed in the specific container sequence 540, only four labeled 3-mer-TCA, TGT, ATG and CAG. 每个标记的3-mer可以在模板500上一个位置且只在一个位置结合。 Each labeled 3-mer and only one location can be combined in one location on the template 500. 由于标记的寡核苷酸520与容器部分540在序列上互补,因此容器部分540中的“A”与寡核苷酸520中的“T”结合,而“G”与“C”结合,反之亦然。 Due to labeled oligonucleotides 520 and the container portion 540 complementary in sequence, and thus "A" 520 to the oligonucleotide container portion 540 of "T" binding, and "G" and "C" binding, and vice versa versa. 探针部分550的序列中的任何变化要求容器部分540序列做相应的变化。 Any change in the sequence of the probe portion 550 of container portion 540 sequences required changes accordingly. 例如,如果探针序列550从AGAA变到AGTA,则容器序列540也必须改变,这是因为探针550中的AGT与容器540中的AGT重叠。 For example, if the probe sequence is changed from 550 to AGAA AGTA, then the container sequence 540 must be changed, because the container probe AGT 550 540 AGT overlap. 一个可能的新的模板500序列示于下面的SEQ ID NO:2。 A possible new template 500 sequence is shown in the following SEQ ID NO: 2.

AGTA AGA ACA TAT GTC(SEQ ID NO:2) AGTA AGA ACA TAT GTC (SEQ ID NO: 2)

[0040] 相应的寡核苷酸520序列为TCT TGT ATA和CAG。 [0040] The corresponding oligonucleotide sequences 520 and TCT TGT ATA CAG. 同样,每个只在容器部分540上的一个位置结合,而且不能与探针部分550结合。 Similarly, only one position on each container part 540 of the binding, but can not bind to the probe portion 550. 对所有可能的4-mer探针序列540进行独特的标记需要18个不同的3-mer标记寡核苷酸520,这比用已知方法产生所有可能的3-mer序列所需的64个标记3-mer 520少得多,已知方法如使用完整的探针文库,通过杂交进行测序。 Uniquely marking all possible 4-mer probe sequence 540 requires 18 different 3-mer oligonucleotide labeled 520, which produce the desired all possible 3-mer sequence markers 64 than with the known methods 3-mer 520 is much less, using known methods such as a complete library of probes, sequencing by hybridization. 在64个可能的3-mer中只用18个也避免了采用可潜在地相互杂交的寡核苷酸520序列所产生的问题。 In the 64 possible 3-mer only with the 18 avoids the problem of using an oligonucleotide sequence 520 can potentially hybridize to each other generated.

[0041] 标记寡核苷酸520(或码组分)可在合成条码530之前提前制备,并进行纯化和储存。 [0041] 520 labeled oligonucleotide (or code components) may be prepared in advance of the synthesis of the bar code 530, and purification and storage. 可以使用给定套的m-mer(m聚体)制备条码530,用于任何所需的探针550序列。 The sleeve may be given m-mer (m mer) prepared bar code 530, 550 for any desired probe sequence. 与已有方法相比,这大大提高了探针550制备的效率,在已有方法中,每个标记探针550分子分开制备而且逐个地标记和纯化。 Compared with the existing methods, which greatly improves the efficiency of the preparation of probes 550, each separately prepared labeled probe molecules 550 in the conventional method and purified and labeled individually. 这里公开的模块体系(modular system)与已知方法相比,表现出标记的高效率。 System disclosed herein module (modular system) compared to known methods, labeled exhibit high efficiency.

[0042] 通常地,将信号(标记)组分附加在核酸链上包括使用标记的核苷酸或合成后标记过程,这两者都会产生问题。 [0042] Generally, a signal (flag) component additionally comprises using the synthetic nucleotides or labeled nucleic acid strand on the marking process, both of which can cause problems. DNA聚合酶一般不能有效地处理标记的核苷酸,将其整合到寡核苷酸520或核酸中。 DNA polymerase generally can not effectively deal labeled nucleotides, for integration into the oligonucleotide or nucleic acid 520. 当要将多个信号组分加到单个核酸链时,整合的效率急剧下降。 To the plurality of signals when the component is added to a single nucleic acid strand, a sharp decline in the efficiency of integration. 由于低的整合效率,具有多于1或2个标记的DNA链需要大量起始物质和标记分子的大量纯化,以使其从未标记或部分标记的分子中分离。 Due to the low integration efficiency, having more than 1 or 2 labeled DNA strand starting materials and a large number of large-scale purification of labeled molecules needed to make it part of the molecule from unlabeled or labeled isolated. 使用这里公开的多个短的标记寡核苷酸520避免了这些问题。 Disclosed herein using a plurality of short labeled oligonucleotide 520 avoids these problems.

[0043] 当条码530分子被设计用于具体的靶分子时,条码530的结构和信号组分被固定,条码530只适合用于一个目的。 [0043] When barcode 530 molecules are designed for a particular target molecule, and the structure of the signal component 530 is fixed to the bar code, the bar code 530 is suitable for a purpose. 如果需要条码530用于其它靶,则必须从头制备每一个。 If needed for other target bar code 530, each must be prepared de novo. 本模块体系使用可提前制备和储存的短的标记寡核苷酸520,用于任何靶,大大提高了灵活性、简便性和产生条码530的速度。 This module system using short labeled oligonucleotides can be prepared in advance and stored 520 nucleotides, for any target, greatly improving the flexibility, simplicity and speed of the bar code 530 is generated. 所需独特标记码组分数目的减少也降低了成本,并提高了检测的效率,这是因为它减少了必须制备和鉴定的可区分标记探针550的数目。 The desired fraction unique marker code also reduces the purpose of reducing costs, and improving the efficiency of detection, since it reduces the number of identification must be prepared and labeled probes 550 may be distinguished.

[0044] 图6说明产生条码的示例性方法,例如上面讨论的条码。 [0044] FIG. 6 illustrates an exemplary method of generating a barcode, for example, bar code discussed above. 例如,通过合成短的寡核苷酸(例如3-mer)以及将标签与寡核苷酸连接或引入已经被标签修饰的核苷酸,可以产生码组分601、602、603、604。 For example, the label and the introduction of oligonucleotides or synthetic oligonucleotides by a short (e.g., 3-mer) has been modified nucleotide tag can be generated code components 601,602,603,604. 与寡核苷酸连接的标签不限于拉曼标签。 Connected to the oligonucleotide tag is not limited to Raman labels. 例如,也可将荧光、纳米颗粒、纳米管、富勒烯和量子点标签连接于寡核苷酸。 For example, it may be a fluorescent, nanoparticles, nanotubes, fullerenes and quantum dot tags attached to the oligonucleotide. 与寡核苷酸的连接模式多种多样。 Oligonucleotide variety of connection modes. 标签可以直接与寡核苷酸相连或通过支链结构相连。 Tags may be directly connected or coupled to the oligonucleotide by a branched structure. 产生用作码组分601、602、603、604的标记寡核苷酸的各种方法是本领域所熟知的。 Various methods used to generate the code components 601,602,603,604 oligonucleotide labeled nucleotides are well known in the art. 可以构建具有扩展探针区域的模板606,其与标记码组分601、602、603、604在序列上互补。 Template 606 may be constructed with an extended region of the probe, which is labeled with a code sequence complementary component 601,602,603,604. 单个地或以混合物的形式,将标记组分601、602、603、604杂交605到模板606上。 Individually or in a mixture, the components 601,602,603,604 marker hybridizes to the template 605 606. 形成的条码607包括具有可检测标签的双链区域和结合到靶分子的单链探针区域。 The barcode 607 is formed having a double-stranded region comprises a detectable label and the probe bound to the single-stranded region of the target molecule.

[0045] 图7说明产生和使用条码的示意图。 [0045] Figure 7 illustrates a schematic view of the generation and use of bar codes. 条码可通过形成模板分子和码组分而产生,如上所述。 Bar code can be produced by forming a template molecule and code components, as described above. 码组分与模板杂交,如上所述,产生条码。 Code component hybridize with a template, as described above, the bar code is generated. 条码产生后,可用于各种目的,例如用于检测样品中的寡核苷酸、核酸或其它靶分子,或者用于测序核酸分子。 After the bar code is generated, it can be used for various purposes, such as for detecting in a sample oligonucleotide, nucleic acid or other target molecules or for sequencing a nucleic acid molecule. 如图7所示,可通过将靶分子重复暴露于包括一个或多个条码的溶液,对核酸靶进行测序。 As shown in FIG 7, by repeated exposure to a target molecule comprising one or more barcode solution, the target nucleic acid sequencing. 条码与靶的杂交表明靶链中互补序列的存在。 Hybridization indicates the presence of the bar code with a target sequence complementary to a target strand. 暴露于不同的条码重复这个过程,表明不同互补序列的存在。 Exposed to different barcode Repeat this process, indicating the presence of different complementary sequences. 如同“鸟枪(shotgun)”测序法中的情形,一些互补序列可重叠。 As "shotgun (Shotgun)" in the case of sequencing, a number of complementary sequences may overlap. 重叠的互补序列可被组合成完整的靶核酸序列。 Overlapping complementary sequences may be combined into a complete target nucleic acid sequence.

[0046] 可将条码引入样品,与靶分子结合,通过任何已知的成象方法进行检测,例如荧光显微法、FTIR(傅里叶变化红外)光谱法、拉曼光谱法、表面等离子体共振和/或电子显微法。 [0046] The bar code may be introduced into the sample, binding to a target molecule, is detected by any known image forming methods, such as fluorescence microscopy, FTIR (Fourier transform infrared) spectroscopy, Raman spectroscopy, surface plasmon resonance and / or electron microscopy.

共价键合的聚合物拉曼标记条码 Raman labels barcode polymer covalently bonded

[0047] 在本发明的某些实施方式中,可产生聚合物拉曼标记条码。 [0047] In certain embodiments of the invention, the polymer may be generated Raman label barcode. 一般而言,聚合物拉曼标记包括拉曼标签直接或通过间隔分子连接其上的骨架部分(backbone moiety)。 Generally, the polymeric Raman label comprises a Raman label or a directly connected portion of its backbone (backbone moiety) through a spacer molecule. 骨架部分可由适于聚合的任何类型单体组成,包括但不限于核苷酸、氨基酸、单糖或任何各种已知的塑料单体,例如乙烯基、苯乙烯、碳酸酯、乙酸酯、乙烯、丙烯酰胺等。 Skeleton portion may be adapted to any type of monomers, including but not limited to, nucleotides, amino acids, monosaccharides, or any of a variety of known plastics monomers, such as vinyl, styrene, carbonate, acetate, ethylene, acrylamide and the like. 聚合物拉曼标记可连接于探针部分上,例如寡核苷酸、抗体、凝集素或适体探针。 Polymeric Raman label can be attached to a probe moiety, such as an oligonucleotide, an antibody, lectin or aptamer probe. 在聚合物骨架由核苷酸单体组成的情况下,与抗体探针的连接会使探针和骨架组分都与不同靶分子结合的可能性最小化。 In the case where the polymer backbone by a nucleotide monomers, with the possibility of connecting the probe causes the probe antibody and skeletal components are bonded to different target molecules is minimized. 可选地,在使用核苷酸单体作为骨架的本发明某些实施方式中,被加入聚合物拉曼标记的核苷酸序列可被设计与靶核酸互补,以使探针功能加入到聚合物拉曼标记中。 Alternatively, in some embodiments of the present invention is used as the backbone of nucleotide monomers, Raman labels are added to the polymer may be designed nucleotide sequence complementary to the target nucleic acid, so that the probe functions added to the polymerization Raman-tag. 由于基于核苷酸的骨架本身产生拉曼发射光谱,有可能干扰所附着拉曼标签的检测,因此在一些实施方式中,使用少产生或不产生拉曼发射信号的骨架组分,以使信号检测最佳化和信噪比最小化。 Since the Raman emission spectrum based on a nucleotide skeleton itself, may interfere with detection of the Raman tag is attached, thus in some embodiments, the use of less or no backbone component to produce Raman emission signal, so that the signal and detection of the optimum SNR is minimized. 以下部分总体上涉及聚合物拉曼标记,并不限于所用单体单元的具体类型。 The following section relates to polymeric Raman labels generally, not limited to specific types of monomer units used.

[0048] 聚合物拉曼标记条码可用于靶分子的检测、鉴定和/或测序,如上所述。 [0048] Polymer may be used to target molecules Raman label barcode detection, identification and / or sequencing, as described above. 探针标记和检测的现有方法具有许多不足。 And detecting labeled probe conventional method has many shortcomings. 例如,与有机荧光标签连接的探针提供高的检测灵敏度,但具有低的多元检测能力。 For example, organic fluorescent label probe connector to provide high detection sensitivity, but has low detection capability polyol. 荧光标签具有宽的发射峰,荧光共振能量转移(FRET)限制了可与单个探针分子连接的不同荧光标签的数量,而自淬灭(self-quenching)减小了荧光信号的量子产生。 Fluorescent tag having a broad emission peaks, fluorescence resonance energy transfer (FRET) limits the number of different fluorescent labels can be connected to a single probe molecule, and the self-quenching (self-quenching) reduces the quantum fluorescent signal is generated. 如果探针包含一个以上类型的发色团,则荧光标签需要多个激发源(excitation source)。 If more than one type of probe comprises a chromophore, a fluorescent label is required a plurality of excitation source (excitation source). 它们也由于光漂白而不稳定。 They are also due to photobleaching and unstable. 潜在的其它类型探针标签是量子点。 Potentially other types of probe label is a quantum dot. 量子点标签是具有多个层的相对较大的结构。 Quantum dot label is a relatively large structure having a plurality of layers. 除了产生复杂外,量子点上的涂层也干扰荧光发射。 In addition to producing complex, the coating layer on the quantum dot fluorescence emission also interfere. 用量子点标签产生的可区分信号的数目也存在限制。 The number of distinguishable signals generated by the quantum dot labels there is a limit. 第三类探针标记由染料浸渍珠子(dye-impregnated bead)组成。 The third probe labeled with a dye impregnated beads (dye-impregnated bead) composition. 这些体积趋于很大,经常大于探针分子的尺寸范围。 These tend to large volume, often greater than the size range of the probe molecule. 染料浸渍珠子的检测是定性的,非定量的。 Detection dye impregnated bead is qualitative, non-quantitative.

[0049] 拉曼标记提供了产生尖光谱峰的优势,允许较多的可区分标记与探针相连。 [0049] The tag provides the advantage of generating a Raman spectrum peak of the tip, allows more distinguishable labeled probe connected. 使用表面增强拉曼光谱(SERS)或类似技术使得检测的灵敏度比得上荧光标签。 Using surface enhanced Raman spectroscopy (SERS) or a similar technique so that the detection sensitivity comparable to a fluorescent label. 示例性的拉曼标签分子的发射光谱示于图8。 Exemplary Raman tag molecule emission spectrum is shown in FIG. 如从图所见,拉曼标签分子提供了可区分光谱的多样性。 As seen from FIGS, the Raman tag molecule provides a distinguishable spectral diversity. 图8表示以下拉曼标签分子的光谱:NBU(寡核苷酸5′-(T)20-脱氧水粉蕈素-T-3′);ETHDA(寡核苷酸5′-(T)-20-(N-乙基脱氧腺苷)-T-3′);BRDA(寡核苷酸5′-(T)-20-(8-溴腺苷)-T-3′);AMPUR(寡核苷酸5′-(T)-20-(2-氨基嘌呤)-T-3′);SPTA(寡核苷酸5′-ThiSS-(T)20-A-3′);和ACRGAM(寡核苷酸5′-acrydite-(G)20-氨基-C7-3′)。 8 shows the Raman spectra of molecules tags: NBU (oligonucleotide 5 '- (T) 20- deoxy nebularine -T-3'); ETHDA (oligonucleotide 5 '- (T) -20 - (N- ethyl-deoxyadenosine) -T-3 '); BRDA (oligonucleotide 5' - (T) -20- (8- bromo-adenosine) -T-3 '); AMPUR (oligonucleotide nucleotide 5 '- (T) -20- (2- aminopurine) -T-3'); SPTA (oligonucleotide 5'-ThiSS- (T) 20-A-3 '); and ACRGAM (oligo nucleotide 5'-acrydite- (G) 20- amino -C7-3 '). 图13表示与核酸光谱本身比较,一个核酸即腺嘌呤的一些核酸类似物的SERS光谱:腺嘌呤;2-F腺嘌呤;4-Am-6-HS-7-脱氮-8-氮杂-腺嘌呤;呋喃甲基氨基嘌呤(kinetin);N6-苯甲酰基-腺嘌呤;DMAA-A;8-氮杂-腺嘌呤;腺嘌呤硫醇和嘌呤衍生物,6-巯基嘌呤。 13 shows a spectral comparison with the nucleic acid itself, SERS spectra of a number of nucleic acids adenine nucleic acid analogs: adenine; 2-F adenine; 4-Am-6-HS-7- deaza-8-aza - adenine; furylmethyl diaminopurine (kinetin); N6- benzoyl - adenine; DMAA-A; 8- aza - adenine; thiol adenine and purine derivatives, 6-mercaptopurine. 表1列出在拉曼光谱中可能使用的其它标签分子。 Table 1 lists other label molecules may be used in the Raman spectrum. 技术人员会认识到,可用的拉曼标签并不限于此处所公开的那些,而可以包括可与探针连接并被检测的任何已知拉曼标签。 In the art will recognize that the available Raman tags is not limited to those disclosed herein, but may include any known Raman label may be coupled to the probe and detected. 在本领域,许多这样的拉曼标签是已知的(参见,例如www.glenres.com)。 In the art, many of Raman labels are known (see, e.g. www.glenres.com).

表1 拉曼标签分子的例子2′,3′-ddA-5′-CE亚磷酰胺2′-脱氧腺苷a-硫代三磷酸(15mM)(2′dATTPaS)2′-氟-腺苷a-硫代三磷酸(10mM)(2′-F-ATTPaS)2′-OMe-A-CE亚磷酰胺2′-OMe-A-Me亚磷酰胺2′-OMe-A-RNA2′-OMe-腺苷a-硫代三磷酸(20mM)(2′-O-Me-ATTPaS)2′-OMe-Pac-A-CE亚磷酰胺2-氨基-dA-CE亚磷酰胺 Table 1 Examples of the Raman tag molecule 2 ', 3'-ddA-5'-CE phosphoramidite 2'-deoxyadenosine a- thiotriphosphate (15mM) (2'dATTPaS) 2'- fluoro - adenosine a- thiotriphosphate (10mM) (2'-F-ATTPaS) 2'-OMe-A-CE phosphoramidite 2'-OMe-A-Me phosphoramidites 2'-OMe-A-RNA2'-OMe - a- thio-adenosine triphosphate (20mM) (2'-OMe-ATTPaS) 2'-OMe-Pac-A-CE phosphoramidite 2-amino -dA-CE phosphoramidite

2-氨基嘌呤核苷a-硫代三磷酸(20mM)(2′-AP-TTPaS)2-F-dA-CE亚磷酰胺3′-A-TOM-CE亚磷酰胺3′-dA-CE亚磷酰胺3′-dA-CPG7-脱氮-腺苷a-硫代三磷酸(1mM)(7-DATTPaS)7-脱氮-dA-CE亚磷酰胺8-氨基-dA-CE亚磷酰胺8-溴-dA-CE亚磷酰胺8-氧-dA-CE亚磷酰胺A-TOM-CE亚磷酰胺A-RNA-TOM-CPG腺苷a-硫代三磷酸(0.5mM)(ATTPaS)Bz-A-CE亚磷酰胺Bz-A-RNA-CPGdA-5′-CE亚磷酰胺dA-5′-CPGdA-CE亚磷酰胺dA-CPG 1000dA-CPG 2000dA-CPG 500dA-高含量-CPG(dA-High Load-CPG)dA-Me亚磷酰胺dA-Q-CPG 500二氨基嘌呤核苷a-硫代三磷酸(0.25mM)(DTTPaS) 2-aminopurine nucleoside a- thiotriphosphate (20mM) (2'-AP-TTPaS) 2-F-dA-CE phosphoramidite 3'-A-TOM-CE phosphoramidite 3'-dA-CE phosphoramidite 3'-dA-CPG7- deaza - a- thio-adenosine triphosphate (1mM) (7-DATTPaS) 7- deaza -dA-CE phosphoramidite 8-amino -dA-CE phosphoramidite 8-bromo -dA-CE phosphoramidite 8--dA-CE phosphoramidite A-TOM-CE phosphoramidite A-RNA-TOM-CPG a- thio adenosine triphosphate (0.5mM) (ATTPaS) Bz-A-CE phosphoramidite Bz-A-RNA-CPGdA-5'-CE phosphoramidite dA-5'-CPGdA-CE phosphoramidite dA-CPG 2000dA-CPG high content -CPG 1000dA-CPG 500dA- ( dA-High Load-CPG) dA-Me phosphoramidites dA-Q-CPG 500 diaminopurine nucleoside a- thiotriphosphate (0.25mM) (DTTPaS)

[0050] 图9说明通过将两个或多个拉曼标记单体单元901、902连接在一起,形成聚合物拉曼标记,从而产生条码的示例性方法。 [0050] Figure 9 illustrates connected together by two or more Raman labels monomeric units 901, 902, to form a polymer Raman labels, exemplary methods to produce barcodes. 该聚合物拉曼标记可以与探针部分相连,以便与靶分子结合以及检测靶分子。 The polymeric Raman label can be attached to the probe portion to a target molecule and detecting binding of target molecules. 聚合物拉曼标记可包括第一单体单元901,其通过共价键906与第二单体单元902连接。 Raman label may comprise a first polymer monomeric units 901, 906 which is connected to the second monomeric unit 902 via a covalent bond. 当需要较大的信号复杂性时,可以连接另外的单体单元903。 When the signal requires a larger complexity, additional monomer units can be connected to 903. 单体单元901、902包括一个或多个拉曼标签部分907a、907b,直接或通过间隔物905连接在骨架909上。 Monomeric units 901, 902 comprises one or more Raman labels portions 907a, 907b, either directly or via a spacer to the backbone 905 909. 间隔物905可包括,例如5个或更多个碳原子。 The spacer 905 may comprise, for example, five or more carbon atoms. 间隔物905的长度可以变化,例如2到30、2到20或3到15个碳原子长。 The length of the spacer 905 may vary, for example, 2 to 30,2 to 20 or 3 to 15 carbon atoms in length. 最有效的间隔物905是柔性的,例如脂肪族碳(如通过氨基己酸)、肽链(如通过赖氨酸侧链连接)或聚乙二醇(如亚磷酰胺)。 The most effective spacer 905 is flexible, such as aliphatic carbon (as determined by amino caproic acid), peptide chains (e.g., lysine side chain connection) or polyethylene glycol (e.g., phosphoramidites). 间隔物905可包括碳、氮、硫和/或氧原子。 The spacer 905 may include carbon, nitrogen, sulfur and / or oxygen atoms. 产生和交联标记单体单元901、902的各种方法是本领域已知的。 Various methods to produce cross-linking and numerals 901 and 902 monomer units are known in the art. 也可从商业渠道获得各种标记单体单元(例如MolecularProbes,Eugene,OR)。 Wide variety of labels may be obtained monomer units (e.g. MolecularProbes, Eugene, OR) are commercially available.

[0051] 如图9所示,可以通过功能基904、908,将一个单体单元901与另一个单体单元902共价连接,形成条码。 [0051] As shown in FIG. 9, the unit 901 is a single bonded to another monomeric unit through functional groups covalently 902 904, 908, the bar code is formed. 功能基904、908可包括,例如生物素、氨基、醛基、硫羟基或本领域所知的其它任何活性基团。 Functional groups 904, 908 may comprise, for example, any other reactive groups known to biotin, an amino group, an aldehyde group, a thiol group, or in the art. 每个单体单元901、902具有至少两个功能基904、908,单体的每一端连接一个。 Each monomer unit having at least two functional groups 901, 902, 904, 908, is connected to each end of a monomer. 在进行交联之前,将一个功能基904、908激活(去保护),以与另一个单体单元901、902连接,而第二个功能基904、908仍被保护,以免受到相互作用或阻断(如通过化学修饰)。 Before crosslinking, 904, 908 will activate a functional group (deprotection), is connected to another monomeric units 901, 902, 904, 908 and the second functional groups are still protected from interactions or hindered off (e.g., by chemical modification). 当激活时,单体单元901、902的每一端都能够与另一单体单元901、902结合。 When activated, each end 901 and 902 monomeric units 901 and 902 can be combined with another monomeric unit. 在各种实施方式中,聚合物拉曼标记包括2到30、4到20或5到15个单体单元901、902(例如,核苷酸、氨基酸、塑性单体等)。 In various embodiments, the polymer comprises a Raman label 2 to 30,4 to 20 or 5 to 15 monomeric units 901, 902 (e.g., nucleotides, amino acids, a plastic monomer). 对由通过共价键906连接在一起的两个单体单元901、902组成的聚合物拉曼标记910的例子,进行了说明。 Raman polymer consisting of two monomer units joined together by covalent bonds consisting of numerals 901, 902 906 910 example, it has been described. 可见拉曼标签907a、907b通过间隔分子905与骨架909相连。 Visible Raman labels 907a, 907b 909 and 905 are connected by a spacer molecule backbone. 单体单元901、902通过共价键906相互连接,在本例中,是通过酰胺键相互连接,该酰胺键例如通过羧基与伯胺基团进行碳二亚胺催化反应而形成。 Monomeric units 901, 902, 906 are connected to each other by a covalent bond, in this embodiment, are connected to each other via an amide bond, the amide bond for example by carbodiimide catalyzed reaction with primary amine groups and carboxyl groups are formed.

[0052] 拉曼标签907a、907b包括一个或多个双键如碳氮双键被考虑在内。 [0052] Raman labels 907a, 907b includes one or more double bonds, such as nitrogen double bond is taken into account. 拉曼标签907a、907b包括带有与环结构相连的侧基的环结构也被考虑在内。 Raman labels 907a, 907b includes a ring structure with side groups attached to the ring structure are also taken into account. 侧基包括但不限于氮原子、氧原子、硫原子和卤素原子以及碳原子和氢原子。 Side groups include, without limitation, a nitrogen atom, an oxygen atom, a sulfur atom and halogen atoms and carbon atoms and hydrogen atoms. 提高拉曼信号检测强度的侧基特别有用。 Pendant groups to improve the Raman signal detection strength are particularly useful. 有效的侧基包括具有共轭环结构的化合物,例如嘌呤、吖啶、罗丹明染料(Rhodamine dye)和花菁染料(Cyanine dye)。 Valid side groups include compounds having a conjugated ring structure, e.g. purine, acridine, rhodamine dyes (Rhodamine dye) and a cyanine dye (Cyanine dye). 考虑聚合物拉曼标记的总极性为亲水性,但可包括疏水侧基。 Consider polymeric Raman label of total polar hydrophilic, but hydrophobic side groups comprise.

[0053] 产生聚合物拉曼标记的示例性方法如图10所示。 Exemplary Method [0053] Raman labels produced polymer is shown in Fig. 用固体支持物1001锚定生长着的聚合物拉曼标记。 1001 solid support anchor the growing polymer Raman label. 该支持物1001可包括,例如有孔玻璃珠、塑料(包括但不限于丙烯酸、聚苯乙烯、苯乙烯和其它物质的共聚物、聚丙烯、聚乙烯、聚丁烯、聚氨酯、特氟隆J等)、多糖、尼龙、硝基纤维素、复合材料、陶瓷、塑料树脂、硅石、硅石基物质、硅、改性硅、碳、金属、无机玻璃、光纤束或其它任何已知类型的固体支持物。 The support 1001 may comprise, for example, of beads, plastic (including but not limited to, acrylic, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon J etc.), polysaccharides, nylon, nitrocellulose, composites, ceramics, plastic resins, silica, silica-based materials, silicon, modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, or any other known type of solid support thereof. 一个或多个接头分子1010(如碳原子链)被连接在支持物1001上。 1010 One or more linker molecules (e.g., carbon atoms in the chain) is connected to the support 1001. 接头分子1010的长度可以变化。 The length of the linker molecule 1010 may vary. 例如,接头1010可以是2-50个原子长度。 For example, the joint 1010 may be a length of 2-50 atoms. 有用的各种接头1010在上面已讨论。 Useful various joints 1010 have been discussed above. 考虑将一个以上长度或类型的接头分子1010连接于固体支持物1001。 Consider the more than one length or type of linker molecule attached to a solid support 1010 1001. 接头1010作为连接点,通过逐步连接单体单元1009而使聚合物拉曼标记生长。 Fitting as a connection point 1010, by progressively connecting the monomer units in the polymer Raman label 1009 growth. 图10显示包括两个单体的聚合物拉曼标记的连接组分1005。 Figure 10 shows the connection component 1005 includes two monomers polymer Raman labeled.

[0054] 要连接的每个单体单元1009包括两个功能基1006、1007,如上所述,在单体单元1009的每一端有一个。 [0054] The monomer units to be connected each comprising two functional groups 1006, 1007 1009, as described above, there is a monomer unit 1009 at each end of. 通过选择性激活单体单元1009前端的功能基1006而实现单体单元1009的加入。 The monomer unit 1009 is realized by selectively activating the monomer unit 1009 1006 distal functional groups. 激活的功能基1006在组分1005的生长端与另一个激活的功能基1004相连。 1006 is connected to the activated functional group end and the other components in the growth of the activated functional group 1005 1004. 化学合成聚合物的方法在本领域是已知的,包括,例如寡核苷酸的亚磷酰胺合成和/或肽的固相合成。 The method of chemical synthesis of polymers are known in the art, including, e.g. phosphoramidite synthesis of oligonucleotides and solid / or phase peptide synthesis. 保护和去保护功能基1004、1006、1007的方法在本领域也是已知的,如在寡核苷酸或肽合成的技术中。 The method of protection and deprotection of functional groups 1004,1006,1007 are also known in the art, such as peptide or oligonucleotide synthesis techniques.

[0055] 可将每个连续的单体单元1009引入溶液中,例如悬浮在乙腈或其它溶剂中。 [0055] each successive monomer may be introduced into the solution unit 1009, for example, suspended in acetonitrile or other solvents. 在第一单体单元1009前端的功能基1006可与接头分子1010结合。 1010 may be combined with a linker molecule in a first functional group monomer units 10061009 front end. 第一单体单元1009与接头分子1010连接后,通过化学处理(如氢氧化铵),将与单体单元1009另一端连接的功能基1007进行去保护,以便结合另一个单体单元1009。 After the first monomer unit 1009 is connected with the linker molecule 1010, the deprotection by chemical treatment (e.g., ammonium hydroxide) and another functional group 1009 connected to the end of 1007 monomer units, in order to bind to another monomeric unit 1009. 要加入的第二单体单元1009可包括激活的功能基1006和保护的功能基1007,以便定向连接单体单元1009。 To add a second monomer unit 1009 may include activating functional group and a protected functional group 1006 1007 1009 in order to orient link monomer units. 在将单体单元1009结合进聚合物拉曼标记的生长组分1005之后,将受保护的功能基1004去保护并加入另一个单体单元1009。 After 1009 monomer units incorporated into the growing polymer Raman label component 1005, and added to the deprotected monomer unit 1009 by another functional group protected in 1004. 继续进行该过程的另一轮,直到产生合适长度的聚合物拉曼标记。 Another round of the process continues until a suitable length polymeric Raman label.

[0056] 考虑可将几个不同的单体单元1009在任何给定的时间加入到固体支持物1001上,以产生不同的聚合物拉曼标记。 [0056] Consider the several 1009 different monomer units at any given time is added to the solid support 1001, to produce different polymeric Raman label. 在后一种情况下,如果合适的话,合成之后,分离不同的聚合物拉曼标记。 In the latter case, if appropriate, after the synthesis, separation of different polymeric Raman label. 聚合物拉曼标记的长度依赖于加入的单体单元1009的数目而发生变化,但是每个聚合物标记将含有两个或更多个单体单元1009。 Polymeric Raman label length depends on the number of added monomer unit 1009 vary, but each tag will be a polymer containing two or more monomer units 1009.

[0057] 在本发明的各种实施方式中,聚合物拉曼标记可包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或更多个拉曼标签1002、1003、1008。 [0057] In various embodiments of the present invention, the polymer may comprise 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 Raman label, 16,17,18,19,20,21,22,23,24,25 or more Raman labels 1002,1003,1008. 与单个聚合物拉曼标记相连的各个拉曼标签1002、1003、1008可以是各不相同的。 Single polymeric Raman label attached to each of the Raman tag 1002,1003,1008 may be different from each other. 可选地,聚合物拉曼标记可包含同一拉曼标签1002、1003、1008的两个或更多个拷贝。 Alternatively, the polymer may contain the same Raman label Raman label two or more copies of 1002,1003,1008. 为最大化可区分聚合物拉曼标记的数目,考虑在多个拉曼标签1002、1003、1008加入单个聚合物拉曼标记的情况下,它们通常是不相同的。 To maximize the number of distinguishable Raman labels polymer, consider a case where a plurality of Raman tags 1002,1003,1008 join individual polymer Raman labels, which are usually not identical. 如上所述,可以将拉曼标签1002、1003、1008直接连接在聚合物拉曼标记1009的骨架1011上或者通过间隔分子连接。 As described above, may be directly connected to a Raman label 1002,1003,1008 polymer backbone 10111009 Raman labels, or via a spacer molecule.

[0058] 聚合物拉曼标签比单体标签提供了更大的光谱区别的多样性,同时提供了拉曼光谱检测的灵敏度。 [0058] Polymer labels Raman difference spectrum provides greater diversity than the monomer tag, while providing detection sensitivity of a Raman spectrum. 使用多个拉曼标签1002、1003、1008与单个聚合物拉曼标记连接使得可以产生很多可区分聚合物拉曼标记。 A plurality of Raman tags 1002,1003,1008 single polymer can produce Raman labels are attached such that many polymers distinguishable Raman label. 由10个不同的可能的标记单体单元1009形成的4-mer聚合物拉曼标记会产生5000个以上的可区分拉曼标记。 4-mer polymer of 10 different Raman label monomer unit 1009 may be formed over the mark 5000 produces distinguishable Raman label. 对于15个不同的标记单体单元1009,会产生30,000个以上的可区分拉曼标记。 For 15 different markers 1009 monomer units, will produce more than 30,000 distinguishable Raman label. 对于只有10到20个不同的标记单体单元1009,可产生50,000个以上的可区分拉曼标记。 For only 10 to 20 different markers 1009 monomer units, more than 50,000 may generate distinguishable Raman label. 由于单体单元1009的大小与核苷酸大约相同(约1000道尔顿),因此4-mer拉曼标记的平均大小大约4000道尔顿。 Since the monomer unit 1009 nucleotide approximately the same size (about 1000 daltons), so the average size of the 4-mer is approximately 4000 Daltons Raman labels. 所以,聚合物拉曼标记允许具有较小空间位阻的探针-靶结合。 Therefore, the polymer having a smaller Raman label probe to allow sterically unhindered - target binding.

[0059] 在本发明的一些实施方式中,加入聚合物拉曼标记的单体单元1009具有与骨架相连的间隔支链,其中另外的活性基团1004、1006、1007与该间隔支链连接。 [0059] In some embodiments of the present invention, the monomer units of the polymer having a Raman interval 1009 marks branched chain attached to the backbone, wherein the additional reactive group is connected to the spacer 1004,1006,1007 branched. 活性基团1004、1006、1007在聚合物合成期间,可以被保护或阻断。 1004,1006,1007 reactive groups during polymer synthesis, can be protected or blocked. 在聚合物合成之后,或在将单体单元1009加入到生长聚合物1005(growing polymer)之后,将拉曼标签1002、1003、1008连接在去保护的间隔支链上。 After the polymer synthesis, or after the monomer unit 1009 is added to the growing polymer 1005 (growing polymer), Raman label attached 1002,1003,1008 deprotection interval branched chain.

[0060] 在本发明的某些实施方式中,如图11A所说明,聚合物拉曼标记1105在没有支持物的情况下产生。 [0060] In certain embodiments of the present invention, as illustrated in FIG. 11A, polymeric Raman label 1105 is generated in the absence of support. 可对拉曼标签1101a、1101b进行化学改变,添加功能基1102a、1102b,例如生物素、氨基、醛基、硫羟基或其它任何类型的活性基团,以产生功能化的拉曼标签(单体单元)1103a、1103b。 Raman labels can 1101a, 1101b chemical change, add functional groups 1102a, 1102b, such as biotin, an amino group, an aldehyde group, a thiol group, or any other type of active group to produce a functionalized Raman label (monomer unit) 1103a, 1103b. 然后将单体单元1103a、1103b进行聚合,产生次级聚合物单元(subpolymeric unit)1104a、1104b,每个都包括预先确定数目的单体单元。 The monomer units 1103a, 1103b polymerization to produce a polymer secondary cell (subpolymeric unit) 1104a, 1104b, each comprising a predetermined number of monomeric units. 将该次级聚合物单元1104a、1104b按预先确定的比例(如1∶1、1∶1、1∶10等)混合在一起,并再进行聚合,产生最终的聚合物拉曼标记1105。 The secondary polymer units 1104a, 1104b proportion (e.g. 1:1,1:1,1:10 etc.) determined in advance were mixed together and then polymerized to produce the final polymer Raman label 1105. 在所示的例子中,聚合物拉曼标记1105包括一种类型单体单元1103a的“n”个拷贝和第二类型单体单元1103b的“m”个拷贝。 In the example shown, the polymeric Raman label 1105 includes one type of monomeric units 1103a "n" "m" copies of copies and a second type of monomer unit 1103b.

[0061] 图11B说明在没有支持物的情况下,产生聚合物拉曼标记1111的可选方法。 [0061] FIG. 11B illustrates the case without support, an alternative method to produce a polymer Raman label 1111. 在此情况下,一个或多个聚合物1109包含活性侧基1112,其连接在延伸出骨架的间隔物上。 In this case, one or more polymer containing reactive pendant groups 1109 1112, which is connected to the spacers extending skeleton. 活性侧基1112可与一个或多个不同的拉曼标签110连接,以形成聚合物拉曼标记1111。 Reactive side groups 1112 may be one or more different Raman labels 110 are connected to form a polymeric Raman label 1111. 活性侧基1112可包括聚赖氨酸,其中聚赖氨酸被处理,以将胺侧基转变为顺丁烯二酰亚胺残基(聚马来酸酐),其可以与HS(硫化氢)功能化的拉曼标签1110反应。 Activity 1112 may include pendant groups polylysine, polylysine which is processed to the amine groups converted to maleimide residues (Poly maleic anhydride), which may be HS (hydrogen sulfide) functionalized Raman labels 1110 reactor. 可选地,侧基1112可包括聚(烯丙胺)的胺基团,其可以与NHS酯功能化的拉曼标签1110反应。 Alternatively, pendant 1112 may include poly (allylamine) amine groups which can react with the NHS ester functionalized Raman label 1110. 侧基1112也可包括丁二酰化聚赖氨酸的羧酸基团或具有氨基或羧酸基的合成寡核苷酸的羧酸基团。 Pendant 1112 may also include succinylated polylysine carboxylic acid group or an amino carboxylic acid group or carboxylic acid group of the synthetic oligonucleotides. 可以例如采用碳二亚胺介导的交联,将羧基化侧基1112连接在拉曼标签1110上。 Crosslinking may be employed, for example, carbodiimide-mediated, carboxyl side groups attached to a Raman label 1112 1110.

[0062] 聚合物骨架可从有机结构形成,例如核酸、肽、多糖和/或化学衍生聚合物的任何组合。 [0062] The polymeric backbone may be formed from the organic structure, such as any combination of nucleic acids, peptides, polysaccharides and / or chemically derivatized polymer. 聚合物拉曼标记1111的骨架可由磷酸二酯键、肽键和/或糖苷键形成。 The polymer backbone by Raman label 1111 phosphodiester bonds, peptide bonds and / or a glycosidic bond is formed. 例如,可用标准的亚磷酰胺化学形成包括DNA链的骨架。 For example, using standard phosphoramidite chemistry comprising forming the backbone of the DNA strand. 形成磷酸二酯连接的骨架的其它方法是已知的,例如聚合酶链反应(PCRTM)扩增。 Other methods of forming phosphodiester backbone connections are known, such as polymerase chain reaction (PCRTM) amplification. 骨架的末端可以具有不同的功能基,例如生物素、氨基、醛基或硫羟基。 End of the backbone may have different functional groups, such as biotin, amino, aldehyde or thiol. 可用这些功能化的基团将两个或多个次级聚合物单元连接在一起。 The available functional groups to link two or more secondary polymer units. 例如,聚合物拉曼标记1111可包括第一单体单元1103a的“m”个拷贝、第二单体单元1103b的“k”个拷贝和第三单体单元的“i”个拷贝。 For example, the polymeric Raman label 1111 may include "m" number of copies of the first monomer unit 1103a, "i" copies 1103b a second monomer unit "k" of copies and the third monomer unit. 聚合物骨架被合成到期望的长度后,可逐个地或同时地引入两个或多个不同的拉曼标签1110,以与活性侧基1112结合,从而产生聚合物拉曼标记。 After the polymer backbone is synthesized to the desired length, it may be introduced individually or simultaneously two or more different Raman tags 1110 to 1112 in combination with the active side groups, to produce a polymer Raman label. 单体单元并不限于拉曼标签1110。 Monomeric units is not limited to a Raman label 1110. 其它的标签,例如荧光、纳米颗粒、纳米管、富勒烯或量子点标签,可与一个或多个单体单元连接,以使聚合物拉曼标记1111多样化。 Other labels, such as fluorescent, nanoparticles, nanotubes, fullerenes, or a quantum dot label, can be connected to one or more monomeric units, to diversify polymeric Raman label 1111. 一般地,单体单元的大多数标签1110将是拉曼标签1110。 Most label 1110 Generally, the monomer unit 1110 is a Raman label. 可加入一个以上聚合物拉曼标记1111,以产生更长的产物。 More than one polymer may be added to a Raman label 1111 to generate longer products.

[0063] 在本发明的某些实施方式中,如图12所示,上面公开的任何的聚合物拉曼标记可与探针1206相连。 [0063] In certain embodiments of the present invention, as shown in FIG. 12, any of the above disclosed polymeric Raman tag 1206 may be connected to the probe. 探针分子1206的例子可包括但不限于寡核苷酸、核酸、抗体、抗体片段、结合蛋白、受体蛋白、肽类、凝集素、底物、抑制物、激活子、配体、激素、细胞因子等。 Examples of probe molecules 1206 can include, but are not limited to oligonucleotides, nucleic acids, antibodies, antibody fragments, binding proteins, receptor proteins, peptides, lectins, substrates, inhibitors, activators, ligands, hormones, cytokines.

聚合物拉曼标记1204的各种示例性结构1201、1202可包括共价连接的单体单元,带有骨架和直接地或通过间隔分子与骨架相连的一个或多个拉曼标签。 Polymeric Raman label various example structures 1201, 1202, 1204 may comprise monomeric units covalently linked to one or more Raman labels with backbone and connected either directly or via a spacer molecule backbone. 聚合物1204可通过接头1205或直接的共价键1205与探针1206连接。 1204 polymer can be direct or via a linker 1205 1205 covalent bond with the probe 1206. 可选地,聚合物拉曼标记1204可间接地,通过连接纳米颗粒1207,与一个或多个探针部分1206相连。 Alternatively, the polymeric Raman label 1204 may be indirectly, by connecting nanoparticles 1207, connected to one or more probes portion 1206. 分子与纳米颗粒交联的各种方法是本领域已知的,可使用任何这样的已知方法。 Molecules and nanoparticles are crosslinked by various methods known in the art, any such known methods. 例如,在存在EDAC(1-乙基-3-(3-二甲基氨基丙基)碳二亚胺)时,通过羧基与氨基的交联来实现。 For example, in the presence of EDAC (1- ethyl-3- (3-dimethylaminopropyl) carbodiimide), is achieved by cross-linking a carboxyl group and an amino group. 如示例性的结构1202所示,可将一个以上的聚合物拉曼标记1204连接在单个纳米颗粒1207上。 As shown in the exemplary structure 1202, may be one or more polymeric Raman label 1204 attached to a single nanoparticle 1207. 然后将纳米颗粒1207连接在一个或多个探针分子1206上。 Nanoparticles 1207 are then connected to one or more probe molecules 1206. 这种结构的优势在于,使用一种聚合物拉曼标记1204,就可以鉴定一个以上的靶分子。 The advantage of this structure is that the use of a polymeric Raman label 1204, can be identified more than one target molecule. 可选地,如果纳米颗粒1207与同一探针分子1206的多个拷贝相连,则可以结合同一靶分子的多个拷贝。 Alternatively, if the nanoparticles are attached to multiple copies of the same probe 1207 to 1206 molecules, you can combine multiple copies of the same target molecule. 其它优势包括捕获靶分子的机会更大,这是因为有更多的探针分子1206与拉曼标记相连,并且由于拉曼标记1202可通过离心、过滤或电泳而被分离,使得在溶液检测应用中,更容易进行自由靶分子与结合拉曼标记的靶分子的分离。 Other advantages include a greater chance of capturing a target molecule, this is because there is more probe molecules attached to Raman labels 1206 and 1202 may be due to the Raman label by centrifugation, filtration or electrophoresis is separated, so that the solution applied in the detection , the more easily separated from the target molecule consisting of a Raman-labeled target molecule.

[0064] 在可选的结构1203中,单体拉曼标签1208直接地或通过间隔分子1205,与纳米颗粒1207相连。 [0064] In an alternative configuration 1203, the monomer directly or Raman tag 1208, the nanoparticles 1207 are connected by a spacer molecule 1205. 一个或多个探针分子直接地或通过间隔物1205与同一纳米颗粒1207相连。 One or more probe molecules are connected directly or via a spacer 1207 1205 same nanoparticles. 这使得连接于探针1206的多个拉曼标签1208的形成不需要聚合物1204的预先合成。 This makes the connection to the probe 1206 is formed of a plurality of Raman tags do not need to previously synthesized polymer 1204 1208. 该结构1203的优势在于,纳米颗粒1207具有较大的表面积,允许结合更多的探针分子1206和拉曼标签1208,并提供分子之间降低的空间位阻。 1203 advantage of this structure is that the nanoparticles have a large surface area 1207, allowing the probe molecules bind more Raman labels 1206 and 1208, and provide reduced steric hindrance between molecules.

[0065] 使用较少的单体单元,可以形成多种聚合物拉曼标记条码。 [0065] fewer monomer units, the polymer may be formed in a variety of Raman labels barcode. 聚合物拉曼标记的产生使得条码的产生具有较大的灵活性和灵敏性,同时使用相对较少的拉曼标签。 Raman label to produce a polymer having a bar code such that greater flexibility and sensitivity, while using a relatively small Raman label.

核酸 Nucleic acid

[0005] 可通过任何标准的技术,制备要测序的核酸分子。 [0005] by any standard technique, the preparation of a nucleic acid molecule to be sequenced. 在一个实施方式中,核酸可以是自然生成的DNA或RNA分子。 In one embodiment, the nucleic acid may be a naturally occurring DNA or RNA molecules. 当使用RNA时,期望将RNA转变成互补的cDNA。 When using RNA, desired to convert the RNA to a complementary cDNA. 事实上,可通过本发明的方法制备和测序任何自然生成的核酸,包括但不限于染色体DNA、线粒体DNA或叶绿体DNA或者信使RNA、不均一核RNA、核糖体RNA或转移RNA。 In fact, by any naturally occurring nucleic acid sequencing and methods of making the present invention, including but not limited to chromosomal DNA, mitochondrial DNA or chloroplast DNA or messenger RNA, heterogeneous nuclear RNA, ribosomal RNA or transfer RNA. 制备和分离各种形式的细胞核酸的方法是已知的(参见,例如,Guide to Molecular Cloning Techniques,eds.Berger和Kimmel,Academic Press,New York,NY,1987;Molecular Cloning:A Laboratory Manual,2nd Ed.,eds.Sambrook,Fritsch和Maniatis,Cold Spring Harbor Press,Cold SpringHarbor,NY,1989)。 The method of preparation and various forms of cells isolated nucleic acid are known (see, e.g., Guide to Molecular Cloning Techniques, eds.Berger and Kimmel, Academic Press, New York, NY, 1987; Molecular Cloning: A Laboratory Manual, 2nd Ed., eds.Sambrook, Fritsch and Maniatis, Cold Spring Harbor Press, Cold SpringHarbor, NY, 1989). 非自然生成的核酸也可以使用公开的方法和组合物进行测序。 Unnatural nucleic acids may be generated using the disclosed methods and compositions were sequenced. 例如,通过标准的扩增技术如聚合酶链反应(PCRTM)扩增制备的核酸可以在本发明的范围内进行测序。 For example, as polymerase chain reaction (PCRTM) amplification of the nucleic acid sequence may be prepared within the scope of the present invention by standard amplification techniques. 核酸扩增的方法在本领域是熟知的。 The method of nucleic acid amplification are well known in the art.

[0006] 核酸可以从多种来源分离,包括但不限于病毒、细菌、真核细胞、哺乳动物以及人类、质粒、M13、λ噬菌体、P1人工染色体(PACs)、细菌人工染色体(BACs)、酵母人工染色体(YACs)和其它克隆载体。 [0006] The nucleic acid may be isolated from a variety of sources, including, but not limited to, viruses, bacteria, eukaryotes, mammals, and humans, the plasmid, M13, λ phage, Pl artificial chromosomes (PACs), bacterial artificial chromosomes (the BACs), yeast artificial chromosomes (YACs) and other cloning vector.

核酸固定化的方法 Nucleic acid immobilization methods

[0007] 在各种实施方式中,通过附着在固体表面上,将核酸分子固定化。 [0007] In various embodiments, by attachment to a solid surface, the immobilized nucleic acid molecule. 核酸分子的固定化可以通过多种方法实现,包括非共价或共价附着在载体或表面上。 Immobilized nucleic acid molecule may be achieved by various methods, including non-covalently or covalently attached to the carrier or surface. 在示例性实施方式中,通过用链霉抗生物素或抗生物素蛋白涂覆固体表面以及结合生物素化的聚核苷酸,实现固定化。 In an exemplary embodiment, the avidin, streptavidin or anti-biotin coated solid surface and binding of biotinylated polynucleotide, is immobilized. 固定化也可以通过用聚-L-Lys或聚L-Lys、Phe涂覆聚苯乙烯、玻璃或其它固体表面,然后用双功能交联剂共价附着氨基或硫氢基修饰的核酸来实现。 Immobilization can also use poly -L-Lys or poly L-Lys, Phe-coated polystyrene, glass or other solid surfaces, and then attached to an amino or sulfhydryl group is modified with a covalent cross-linker to achieve nucleic acid . 用氨基硅烷,可将胺残基引入到表面上。 Aminosilane, amine residues can be introduced onto the surface.

[0008] 固定化可通过将5′-磷酸化核酸直接共价附着在化学修饰的聚苯乙烯表面而进行。 [0008] The immobilization can be obtained by 5'-phosphorylated nucleic acid is covalently attached directly carried out in chemically modified polystyrene surface. 核酸和固体表面之间的共价键通过与水溶性碳二亚胺缩合而形成。 Covalent bond between the solid surface and the nucleic acids formed by the condensation of a water-soluble carbodiimide. 这种方法促进了核酸通过其5′-磷酸主要进行的5′-附着。 This method facilitates the attachment of nucleic acid through 5'-5'-phosphate which is mainly performed.

[0009] 通常先使玻璃表面硅烷化,然后用碳二亚胺或戊二醛活化,将DNA结合在玻璃上。 [0009] generally to the glass surface is then silanized with carbodiimide or glutaraldehyde activation, DNA binding to the glass. 可选的过程可使用试剂如3-环氧丙氧基丙基三甲氧基硅烷或氨基丙基三甲氧基硅烷(APTS),以及通过氨基接头连接的DNA,该氨基接头在DNA合成过程中被加入在分子的3′或5′末端。 The process may optionally using reagents such as 3-glycidoxypropyl trimethoxysilane or aminopropyl trimethoxy silane (the APTS), and a DNA linker via an amino group, the amino group in the linker DNA synthesis is molecule is added to the 3 'or 5' end. 使用紫外线辐射,可将DNA直接与膜结合。 Ultraviolet radiation, DNA can be bound directly to membranes. 固定化核酸的其它方法是已知的。 Other methods of immobilized nucleic acids are known.

[0010] 用于固定化核酸的表面类型没有限制。 [0010] for the type of surface immobilized nucleic acids is not limited. 在各种实施方式中,固定化表面可以是磁珠子、非磁性珠子、平面、打点表面(pointed surface)或包括几乎任何物质的其它任何构型的固体表面,只要该材料允许核酸与探针文库杂交。 In various embodiments, the immobilization surface can be magnetic beads, non-magnetic beads, flat surfaces RBI (pointed surface), or any other configuration comprising a solid surface of virtually any material, as long as the material allows a library of nucleic acid probe hybridization.

[0011] 双功能交联剂可用于许多实施方式。 [0011] Bifunctional cross-linking agent may be used in many embodiments. 示例性的交联剂包括戊二醛、双功能环氧乙烷、乙二醇二缩水甘油醚和碳二亚胺,例如1-乙基-3-(-二甲基氨基丙基)碳二亚胺。 Exemplary crosslinking agents include glutaraldehyde, bifunctional oxirane, ethylene glycol diglycidyl ether, and carbodiimides, such as 1-ethyl-3 - (- dimethylaminopropyl) carbodiimide imide.

[0012] 在某些实施方式中,捕获寡核苷酸(capture oligonucleotide)可与表面结合。 [0012] In certain embodiments, the capture oligonucleotides (capture oligonucleotide) can be bound to the surface. 该捕获寡核苷酸将与核酸模板的特定核酸序列杂交。 The capture oligonucleotides hybridize to a nucleic acid sequence specific nucleic acid template. 通过限制酶消化、内切核酸酶活性、提高的温度、降低的盐浓度或这些和相似方法的组合,可将核酸从表面释放。 By restriction enzyme digestion, endonuclease activity, elevated temperature, decreasing salt concentration or a combination of these and similar methods, a nucleic acid can be released from the surface.

蛋白质的纯化 Purification of proteins

[0013] 在某些实施方式中,对蛋白质或肽进行分离或纯化。 [0013] In certain embodiments, the protein or peptide to be isolated or purified. 在一个实施方式中,这些蛋白质用于产生抗体,以便用任何所述条码(如聚合物拉曼标记)进行标记。 In one embodiment, these proteins for generating antibodies, for use in any of the bar code tag (e.g., polymeric Raman label). 蛋白质纯化技术是本领域技术人员所熟知的。 Protein purification techniques are well known to the skilled person. 这些技术包括,在一个水平上,将细胞、组织或器官匀浆化和粗分级分离为多肽组分和非多肽组分。 These techniques involve, at one level, the isolated cell, tissue or organ homogenization and crude fractionation of the polypeptide component and a non-polypeptide component. 用层析和电泳技术对感兴趣的蛋白质或多肽进行进一步的纯化,以实现部分或完全的纯化(或纯化至同质性)。 Further purification of the protein or polypeptide of interest using chromatographic and electrophoretic techniques to achieve complete purification or partially (or purification to homogeneity). 特别适合用于制备纯肽的分析方法是离子交换色谱法、凝胶排阻色谱法、HPLC(高效液相色谱法)、FPLC(AP Biotech)、聚丙烯酰胺凝胶电泳法、亲和色谱法、免疫亲和色谱法和等电点聚焦法。 Analysis method is particularly suitable for the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, HPLC (high performance liquid chromatography), FPLC (AP Biotech), polyacrylamide gel electrophoresis, affinity chromatography , immunoaffinity chromatography and isoelectric focusing method. 通过亲和色谱法纯化受体蛋白的例子在美国专利US 5,206,347中公开,在此引入其全部内容作为参考。 Examples of receptor protein purification by affinity chromatography is disclosed in U.S. Patent No. US 5,206,347, incorporated herein by reference in its entirety. 纯化肽的最有效的方法之一是快速性能液相色谱法(fast performance liquidchromatography)(AKTA FPLC)或甚至HPLC。 One of the most efficient method of purifying peptides is fast performance liquid chromatography (fast performance liquidchromatography) (AKTA FPLC) or even HPLC.

[0014] 纯化的蛋白质或肽被有意称为组合物,是可以与其它组分分离的,其中蛋白质或肽相对于它的自然可得的状态,可被纯化到任何程度。 [0014] The purified protein or peptide is intentionally referred to as compositions, it can be separated from other components, wherein the protein or peptide with respect to its natural state available, can be purified to any degree. 因此,分离的或纯化的蛋白质或肽也被指脱离其自然生成环境的蛋白质或肽。 Thus, an isolated or purified protein or peptide is also alleged departing from naturally occurring proteins or peptides environment. 一般地,“纯化的(purified)”指已经接受分级分离而除去其它各种组分的蛋白质或肽组合物,并且其组成成分基本保持其表达的生物学活性。 Generally, "purified (Purified)" which has been accepted and the fractionation protein or peptide composition to remove various other components, and which composition substantially maintaining the biological activity of its expression. 当使用术语“基本纯化的(substantially purified)”时,该名称指组合物中蛋白质或肽形成该组合物的主要组分,例如在组合物中,蛋白质构成约50%、约60%、约70%、约80%、约90%、约95%或更多。 When the term "substantially purified (substantially purified)" when the name refers to the major component of the composition of a protein or peptide composition is formed, for example, in the composition, the protein comprises about 50%, about 60%, about 70 %, about 80%, about 90%, about 95% or more.

[0015] 量化蛋白质或肽的纯化程度的各种方法是本发明所述领域的技术人员知晓的。 [0015] The quantization level of the protein or peptide is purified by various methods is the field of the invention known to the art. 这些包括,例如,测定活性组分的比活性,或通过SDS/PAGE分析法评估组分中多肽的含量。 These include, for example, determining the specific activity of the active ingredient, content of the component or the polypeptide is assessed by SDS / PAGE analysis. 评估一个组分纯度的优选方法是计算该组分的比活性,将它与初始提取物的比活性比较,以及由此计算其中的纯度,用“纯化倍数(-foldpurification number)”进行评估。 Evaluation method of a preferred component of the component purity is calculated specific activity, the specific activity of the initial extract it was compared, and wherein the purity of the thus calculated, with the "purification factor (-foldpurification number)" for evaluation. 用于表示活性量的实际单位当然取决于纯化之后所选用的具体测试方法,以及表达的蛋白质或肽是否表现出可检测的活性。 The actual units used to represent the amount of activity of course depends on the specific test method after purification chosen, and whether the expressed protein or peptide exhibits a detectable activity.

[0016] 适合用于蛋白质纯化的各种技术是本领域技术人员所熟知的。 [0016] Various techniques suitable for protein purification are well known to the skilled person. 这些包括,例如,硫酸铵沉淀、PEG、抗体和类似技术或通过加热变性,然后进行:离心;色谱法步骤,例如离子交换色谱法、凝胶过滤色谱法、反相色谱法、羟基磷灰石色谱法及亲和色谱法;等电点聚焦;凝胶电泳;以及这些和其它技术的结合。 These include, for example, precipitation with ammonium sulfate, PEG, antibodies and similar techniques or by heat denaturation, followed by: centrifugation; chromatography steps such as ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, hydroxyapatite chromatography and affinity chromatography; isoelectric focusing; gel electrophoresis; and a combination of these and other techniques. 如本领域通常已知的,相信,进行各种纯化步骤的次序可以改变,或者某些步骤可以略去,而仍形成制备基本纯化的蛋白质或肽的合适方法。 As is generally known in the art, it is believed the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still suitable methods for preparing substantially purified protein or peptide is formed.

[0017] 一般不要求蛋白质或肽总是以它的最纯化的状态被提供。 [0017] does not generally require the protein or peptide always be provided in its most purified state. 事实上,在某些实施方式中使用较低基本纯化的产物被考虑在内。 In fact, the use of substantially purified product was lower in certain embodiments be considered. 部分纯化可以通过采用结合起来的较少纯化步骤来完成,或者通过采用相同总体纯化方案的不同形式来完成。 Partial purification may be accomplished by using fewer purification steps in combination, or work through different forms of the same general purification scheme. 例如,可以理解,用HPLC设备进行的阳离子交换柱色谱法比采用低压色谱系统的相同技术,一般将产生较大“倍数(-fold)”的纯化。 For example, it is understood, by HPLC cation exchange column chromatography apparatus with the same ratio of a low pressure chromatography system technology, purified large "fold (-fold)" will generally be produced. 表现出较低程度的相对纯化的方法在蛋白产物的总回收上,或者在保持表达蛋白的活性上具有优势。 The method exhibits a relatively low degree of purification have advantages in total recovery of protein product, or in maintaining the activity of protein expression.

[0018] 亲和色谱法是依赖于被分离的物质与其特定结合的分子之间特异性亲和力的色谱方法。 [0018] Affinity chromatography is a chromatographic method relies on the specific affinity between a substance to be separated molecules to their specific binding. 这是受体-配体型的相互作用。 This is a receptor - ligand type interaction. 通过将结合对之一与不溶性基质共价偶联,合成柱材料。 By binding to one insoluble matrix covalently coupled, synthesis column material. 然后柱材料能够特定地从溶液中吸收物质。 Column material is then able to absorb a specific substance from the solution. 通过将条件改变到结合不发生的条件(例如,改变pH、离子强度、温度等),进行洗脱。 By binding conditions were changed to conditions does not occur (e.g., altered pH, ionic strength, temperature, etc.), for elution. 基质应当是它本身不明显吸收分子的物质以及具有较宽的化学、物理和热稳定性的物质。 Medium should be obvious molecules of the substance itself, and has a broad absorption chemical, physical and thermal stability of the material. 配体应当以不影响它的结合性质的方式进行偶联。 Ligand should not affect its binding properties coupled manner. 配体也应当提供较为紧密的结合作用。 Ligand should also provide relatively tight binding. 而且,应当可能在不破坏样品或配体的情况下洗脱该物质。 Further, it should be possible to elute the substance without destroying the sample or the ligand.

[0019] 蛋白质或肽可用本领域技术人员已知的任何技术制备,包括通过标准的分子生物学技术表达蛋白质、多肽或肽;从自然来源分离蛋白质或肽;或者化学合成蛋白质或肽。 [0019] proteins or peptides prepared by any known to those skilled in the art can be used, including standard molecular biological techniques by expression of proteins, polypeptides or peptides; isolated from a natural source of protein or peptide; or chemically synthesized protein or peptide. 对应于各种基因的核苷酸和蛋白质、多肽和肽序列已经被公开,并且可以在本领域普通技术人员知晓的计算机化数据库中找到。 Corresponding to the nucleotide and protein, polypeptide and peptide sequences of various genes have been disclosed, and can be found in known to those of ordinary skill in computerized databases. 一个这种数据库是National Center for Biotechnology Information's GenBank and GenPept数据库(http://www.ncbi.nlm.nih.gov/)。 One such database is the National Center for Biotechnology Information's GenBank and GenPept databases (http://www.ncbi.nlm.nih.gov/). 使用这里公开的技术或本领域普通技术人员知晓的技术,可以扩增和/或表达已知基因的编码区域。 Or using the disclosed techniques known to those of ordinary skill in this art, can be amplified and / or expression of the coding region of known genes. 替代地,蛋白质、多肽和肽的各种商业制备物是本领域技术人员已知的。 Alternatively, the proteins, polypeptides and peptides of various commercial preparation is known to the skilled person.

肽模拟物(peptide mimetics) Peptide mimetics (peptide mimetics)

[0020] 本发明制备多肽的另一种方法是使用用于单克隆抗体生产的肽模拟物(peptide mimetics)。 [0020] Another method for preparing polypeptide of the invention is the use of monoclonal antibodies for the production of peptide mimetics (peptide mimetics). 模拟物(mimetics)是含肽的分子,其模拟蛋白质二级结构的元件。 Mimetic (mimetics) are peptide-containing molecules, which mimic elements of protein secondary structure. 参见,例如,Johnson等,“Peptide Turn Mimetics”,BIOTECHNOLOGYAND PHARMACY中,Pezzuto等,Eds.,Chapman and Hall,New York(1993),在此引入作为参考。 See, e.g., Johnson, etc., "Peptide Turn Mimetics", BIOTECHNOLOGYAND PHARMACY in, Pezzuto the like, Eds., Chapman and Hall, New York (1993), incorporated herein by reference. 使用肽模拟物的基本原理在于,蛋白质的肽骨架主要以这样的方式朝向氨基酸侧链而存在,以促进分子相互作用,例如抗体和抗原的相互作用。 The basic principle of the use of peptide mimetics is that the peptide backbone of proteins exists chiefly in such a manner toward the side chains of amino acids present to facilitate molecular interactions, such as antibodies and antigen interaction. 预期肽模拟物允许分子相互作用,如同天然分子一样。 Peptide mimetic is expected to permit molecular interactions, as the same natural molecule. 这些原理可用于工程设计第二代分子,其具有这里公开的靶向肽(targeting peptides)的许多自然性质,但是具有改变的和甚至改进的特性。 These principles can be used in engineering design second generation molecule having a targeting peptide (targeting peptides) disclosed herein many natural properties, but with altered and even improved characteristics.

融合蛋白 The fusion protein

[0021] 本发明的其它实施方式涉及融合蛋白。 [0021] Other embodiments of the present invention relates to fusion proteins. 这些分子一般具有靶向肽的全部或基本部分,其在N-或C-末端连接到第二多肽或蛋白质的所有或部分上。 These molecules generally have all or a substantial portion of a targeting peptide, which is connected to all or a portion of a second polypeptide or protein at the N- or C- terminus. 例如,融合可以使用来自其它物种的前导序列,以允许蛋白质在异源宿主中重组表达。 For example, fusion may be used leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. 另一种有用的融合包括添加免疫学活性结构域,例如抗体表位,以促进融合蛋白的纯化。 Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. 在融合接点处或附近加入切割位点将促进纯化后额外多肽的去除。 Remove additional polypeptide fused or in the vicinity of the junction point cleavage site was added to facilitate purification. 其它有用的融合包括功能结构域的连接,例如酶的活性位点、糖基化结构域、细胞靶向信号或跨膜区域。 Other useful fusions include linking of functional domains, such as an enzyme active sites, glycosylation domains, cellular targeting signals or transmembrane regions. 在某些实施方式中,融合蛋白包括与治疗用蛋白质或肽连接的靶向肽。 In certain embodiments, the fusion protein comprising a targeting peptide and therapeutic protein or peptide linked. 考虑在本发明的范围内,事实上,可将任何蛋白质或肽加入包括靶向肽的融合蛋白。 Considered within the scope of the present invention, in fact, it may be any protein or peptide to a fusion protein comprising a targeting peptide. 产生融合蛋白的方法是本领域技术人员熟知的。 The method of generating fusion proteins are well known to the skilled person. 这种蛋白质可以通过如下途径产生,例如:使用双功能交联剂的化学附着;完整融合蛋白的从头合成;或者将编码靶向肽的DNA序列与编码第二个肽或蛋白质的DNA序列连接,然后表达完整的融合蛋白。 Such proteins may be produced by the following route, for example: using a chemical cross-linker is attached; de novo synthesis of the complete fusion protein; or connect a DNA sequence encoding a DNA sequence encoding the targeting peptide to a second peptide or protein, then the full expression of the fusion protein.

合成的肽 Synthetic peptides

[0022] 由于它们的相对较小的体积,真菌选择方法鉴定的肽可以根据传统技术,在溶液中合成,或者在固体支持物上合成。 [0022] Because of their relatively small size, the peptides identified fungal selection method according to the conventional art, synthesized or synthesized on a solid support in solution. 各种自动化合成仪是商业可得的,并且可根据已知方案使用。 Various automated synthesizers are commercially available and may be used in accordance with known protocols. 参见,例如,Stewart和Young,(1984);Tam等,(1983);Merrifield,(1986);以及Barany和Merrifield(1979),在此引入每一个作为参考。 See, e.g., Stewart and Young, (1984); Tam et, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. 用这些方法,可以容易合成短的肽序列,通常为约6至高达约35到50个氨基酸。 With these methods, it can be readily synthesized short peptide sequences, usually from about 6 up to about 35 to 50 amino acids. 可选地,可使用重组DNA技术,其中编码本发明中肽的核苷酸序列被插入表达载体、被转化或转染到合适的宿主细胞中,并在适于表达的条件下进行培育。 Alternatively, recombinant DNA technology may be used, according to the present invention wherein the nucleotide sequence encoding the peptide is inserted into an expression vector, transformed or transfected into a suitable host cell, and incubated under conditions suitable for expression.

示例性应用核酸测序 Exemplary nucleic acid sequencing applications

[0023] 在具体的实施方式中,可用这里公开的方法所形成的条码测序靶核酸分子。 [0023] In a specific embodiment, the bar code can be used sequencing methods disclosed herein formed by the target nucleic acid molecule. 通过杂交测序的方法是本领域已知的。 The method of sequencing by hybridization are known in the art. 可以使一个或多个包括已知序列的探针的标记条码杂交到靶核酸序列。 It can be made to one or more target nucleic acid sequences comprising a bar code labeled hybridization probe of known sequence. 标记条码与靶的结合表明靶链中互补序列的存在。 Barcode binding of the labeled target to the target strand indicative of the presence of complementary sequences. 可使多个标记条码与靶分子同时杂交并同时检测。 A plurality of bar code tag can simultaneously hybridize to a target molecule and detected simultaneously. 在可选的实施方式中,结合的探针可以被鉴定与各个靶分子相连,或者可选地,可以使具体靶分子的多个拷贝与探针序列的重叠集合同时结合。 In an alternative embodiment, the binding probe may be connected to each identified target molecule, or alternatively, a plurality of overlapping sets of copies can be made with the probe sequence specific target molecule binding at the same time. 对各个分子,可以例如使用已知的与检测方式结合的分子梳理技术(molecular combing technique)进行扫描。 Each molecule, for example, using a known detection method in combination with the carding molecular techniques (molecular combing technique) scanning. (参见,例如,Bensimon等,Phys.Rev.Lett.74:4754-57,1995;Michalet等,Science 277:1518-23,1997;美国专利US 5,840,862;US 6,054,327;US 6,225,055;US 6,248,537;US 6,265,153;US 6,303,296和US 6,344,319。) (See, e.g., Bensimon et, Phys.Rev.Lett.74: 4754-57,1995; Michalet et, Science 277: 1518-23,1997; U.S. Patent No. US 5,840,862; US 6,054,327; US 6,225,055; US 6,248,537; US 6,265,153 ; US 6,303,296 and US 6,344,319).

[0024] 给定的靶核酸与完全覆盖靶序列的邻接探针序列杂交是不可能的。 [0024] a given target nucleic acid probe hybridizes to a sequence adjacent to completely cover the target sequence is not possible. 更合适地,将靶的多个拷贝与多个标记寡核苷酸进行杂交,并从每一个,收集部分序列数据。 More suitably, a plurality of copies of the target with a plurality of labeled oligonucleotide hybridization, and from each of a sequence of data collection portion. 采用公共可得的鸟枪序列汇编程序(shotgun sequence compilationprogram),可将部分序列编译成完整的靶核酸序列。 Using publicly available shotgun sequence assembler (shotgun sequence compilationprogram), a partial sequence may be compiled into a complete target nucleic acid sequence. 也可从靶分子的群体编译部分序列,该靶分子群体被允许例如在溶液相中同时与一文库的条码探针进行结合。 It may be compiled from the partial sequence of the target molecule population, for example, the target molecule population is allowed to simultaneously be combined with a library barcode probe in solution phase.

靶分子检测、鉴定和/或量化 Target molecule detection, identification and / or quantification of

[0025] 在某些实施方式中,通过与条码结合,可以检测、鉴定和/或量化样品中的靶分子。 [0025] In certain embodiments, the binding by the barcode can be detected, identified and / or quantified in a sample of target molecules. 设计与具体靶结合的标记条码可按如上所述进行制备。 Design of specific target binding barcode label can be prepared as described above. 靶不限于核酸,但也可包括蛋白质、肽类、脂类、糖类、糖脂、糖蛋白或其它可以为其制备具体探针的任何可能的靶。 It is not limited to the target nucleic acid, but may also include proteins, peptides, lipids, carbohydrates, glycolipids, glycoproteins or any other potential target can be prepared for a particular probe. 如上所述,可将抗体或适体结合进条码,并用于鉴定可为其制备适体或抗体的任何靶标。 As described above, antibody or aptamer may be incorporated into the bar code, and may be used to identify any target for which an aptamer or antibody preparation. 可以同时测试样品中多个靶的存在,这是因为每个条码可以被可区分地标记和检测。 Present in the sample can be tested at the same time a plurality of targets, because each barcode may be labeled and detected distinguishable. 可采用标准的技术进行靶的量化,这些技术在光谱分析中是熟知的。 Standard techniques may be employed to quantify the target, these techniques are well known in spectroscopic analysis. 例如,通过测量结合条码的信号强度以及与从已知量条码标准得到的校正曲线进行比较,可以测定与标记条码结合的靶的量。 For example, compared with a calibration curve obtained from known standard bar code by measuring the amount of binding of the signal strength and bar code, the amount of bound labeled with bar code of the target can be determined. 这些量化方法完全在本领域的常规技术之内。 These quantitative methods are well within the routine skill in the art.

阵列化学 An array of chemical

[0026] 可以将具有不同化学功能性(例如不同结合特异性)的珠子(例如微球)混合在一起。 [0026] may have a different chemical functionality (e.g. different binding specificities) beads (e.g. microspheres) are mixed together. 采用光学可询编码方案(optically interrogatable encodingscheme)(“光学签名(optical signature)”),可以实现鉴定每个珠子功能性的能力。 Inquiry using optically encoding scheme (optically interrogatable encodingscheme) ( "optical signature (optical signature)"), ability to identify the functionality of each bead can be achieved. 例如,使用如上所述的聚合物拉曼标记,可以产生光学签名。 For example, a polymer as described above, Raman labels, optical signature may be generated. 基质,例如芯片或微量滴定板,可包括含有独立位点的有图案表面,所述独立位点与各个珠子相结合。 Matrix, such as a chip or microtiter plates, may comprise a patterned surface containing a separate site, the site with the respective independent beads combined. 这使得探针(即,核酸、适体或抗体)的合成可以与其在阵列上的定位分离开来。 This makes the probes (i.e., a nucleic acid, aptamer or antibody) and its synthesis can be positioned on the array separated. 探针可被合成、与珠子相连而且珠子随机地分布在有图案表面上。 Probes can be synthesized, coupled to the beads and the beads are randomly distributed on a patterned surface. 由于珠子首先用光学签名编码,因此形成的阵列(array)稍后可被“译码”。 Since the array (Array) with a first optical signature encoded beads thus formed can be "decoded" later. 就是说,阵列上各个位点位置与位于该具体位点的珠子或探针之间的关系可以被得到。 That is, the positional relationship between the respective sites located at the particular site with the probe beads or the array can be obtained. 由于珠子随机分布在阵列上,与产生阵列的原位合成或定位技术(spotting technique)相比,这形成了快速及低成本的方法。 Because the beads are randomly distributed on the array, as compared with in situ synthesis techniques or positioning arrays (spotting technique), which forms a fast and low-cost method.

[0027] 阵列组合物可包括至少第一基质,其具有包括各个位点的表面。 [0027] Array compositions may include at least a first substrate having a surface comprising individual sites. 阵列大小取决于阵列的最终用途。 Array size depending on the end use of the array. 可形成包含大约2个不同介质(agent)(即不同珠子)到上百万个不同介质的阵列。 It may form comprising about two different media (Agent) (i.e. different beads) to an array of millions of different media. 一般地,阵列包括两个不同珠子到十亿个或更多,这取决于珠子和基质的尺寸。 Generally, the array comprising two beads of different sizes to one billion or more, depending on the beads and the substrate. 因此,可形成很高密度、高密度、中密度、低密度或很低密度的阵列。 Thus, the formation of high density, high density, medium density, low density or very low density arrays. 很高密度阵列的一些范围是每阵列为约10,000,000到约2,000,000,000个位点。 Some high density arrays range per array is about 10,000,000 to about 2,000,000,000 sites. 高密度阵列的范围是约100,000到约10,000,000个位点。 High density arrays range from about 100,000 to about 10,000,000 sites. 中密度阵列的范围是约10,000到约50,000个位点。 Density arrays range from about 10,000 to about 50,000 sites. 低密度阵列一般少于10,000个位点。 Low density array of generally less than 10,000 sites. 很低密度阵列少于1,000个位点。 An array of very low density less than 1,000 sites.

[0028] 在本发明的一些实施方式中,可使用多个基质,具有不同或相同的组合成分。 [0028] In some embodiments of the present invention, a plurality of substrates can be used, with different or the same combination of ingredients. 因此例如,大阵列可包括多个较小的基质。 Thus for example, large arrays may comprise a plurality of smaller substrates. “基质(substrate)”或“固体支持物(solid support)”意指可以被修饰而包含不连续的各个位点的任何材料,其适应于至少一种检测方法,所述位点适合于珠子的附着或结合。 "Matrix (Substrate)" or "solid support (solid support)" means that may be modified to comprise any material discontinuity each site, which is adapted to at least one detection method, the site is adapted to beads attachment or bonding. 一般地,基质允许光学检测而不会感到对信号发射有干扰。 Generally, the matrix allowing optical detection without interference to the signal transmission feel.

[0029] 位点包括图案,即规则的设计或构型,或者位点为随机分布。 [0029] The site includes a pattern, i.e. a regular design or configuration, or randomly distributed sites. 可使用规则图案的位点,以便位点集中在XY坐标平面上。 Site using a regular pattern of sites to focus on the XY coordinate plane. 基质表面可以被修饰,以允许微球在各个位点附着。 The substrate surface may be modified to allow the microspheres attached at each site. 因此,可修饰基质表面,以便形成不连续位点,其只有单个结合的珠子。 Thus, the substrate surface may be modified, so as to form a discontinuous site, which binds only a single bead. 在一个实施方式中,修饰基质表面,使其包含孔,即底物表面的凹陷。 In one embodiment, the modified substrate surface, so that it contains pores, i.e. the concave surface of the substrate. 这可以使用各种已知技术来进行,包括但不限于照相平板印刷术(photolithography)、冲压技术(stamping technique)、塑型技术(molding technique)以及微蚀刻技术(microetching technique)。 This can be performed using various known techniques, including but not limited to photolithography (photolithography), stamping (stamping technique), molding techniques (molding technique) and microetching techniques (microetching technique). 正如本领域技术人员理解,所用技术取决于基质的组成和形状。 As understood by those skilled in the art, the technique used depends on the composition and shape of the matrix. 可选地,修饰基质表面,使其包括化学衍生的位点,该位点可以用于将微球和/或珠子附着在基质的不连续位置上。 Alternatively, the modified substrate surface, chemically derivatized to include a site, the site may be used for the microspheres and / or beads attached to the discrete locations on the substrate. 可以采用加入化学功能基的图案,例如氨基、羧基、氧基和硫羟基,以便共价附着微球,微球一般包含相应的活性功能基或接头分子。 Chemical functionality group can be added using a pattern, such as amino, carboxyl, and thiol groups, covalently attached to microspheres, the microspheres generally comprise a respective active functional group or a linker molecule.

[0030] 合适的珠子组合物包括用于肽、核酸和有机部分合成中的那些,包括但不限于塑料、陶瓷、玻璃、聚苯乙烯、甲基苯乙烯、丙烯酸聚合物、顺磁物质、氧化钍溶胶、碳石墨、二氧化钛、乳胶或交联葡聚糖如Sepharose(琼脂糖)、纤维素、尼龙、交联微团以及Teflin,都可以使用。 [0030] Suitable bead compositions include a peptide, nucleic acid and organic moiety synthesis, and include but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, oxide thorium sol, carbon graphite, titanium dioxide, latex or cross-linked dextran such as Sepharose (Sepharose), cellulose, nylon, cross-linked micelles and Teflin, can be used. 珠子大小范围可以从纳米即100nm到毫米即1mm,珠子可以为约0.2微米到约200微米以及从约0.5到约5微米,但在一些实施方式中可以使用更小的珠子。 Bead sizes may range from nanometers to millimeters i.e. 100nm i.e. 1mm, the beads may be from about 0.2 microns to about 200 microns, and from about 0.5 to about 5 microns, in some embodiments smaller beads may be used.

[0031] 可使用组合物检测具体靶分析物的存在,例如核酸、寡核苷酸、蛋白质、酶、抗体或抗原。 [0031] The composition may be used to detect the presence of a particular target analytes, such as nucleic acid, oligonucleotide, protein, enzyme, antibody or antigen. 也可使用组合物筛选生物活性物质(bioactive agent),即药物候选物,筛选其与具体靶的结合,或用于检测污染物之类的物质。 Screening may also be used in combination with the biologically active substance composition (bioactive agent), i.e. drug candidates, screened for their binding to a particular target or class of substance for the detection of pollutants. 如上所述,可以为其设计探针部分如肽、蛋白质、寡核苷酸或适体的任何分析物可以和所公开的条码结合使用。 As described above, it is possible to design a probe portion such as a peptide, protein, oligonucleotide or aptamer of any analyte and the bar code can be used in conjunction disclosed.

[0032] 生物活性物质可以从各种来源获得,包括合成或天然化合物的文库。 [0032] The biologically active substance may be obtained from a variety of sources, including libraries of synthetic or natural compounds. 例如,许多方法可以用于随机和定向合成各种有机化合物和生物分子,包括随机化寡核苷酸的表达。 For example, many methods may be used for random and directed synthesis of a variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. 可选地,细菌、真菌、植物和动物提取物形式的天然化合物文库是可用的或容易制备。 Alternatively, libraries of natural compounds in bacterial, fungal, plant and animal extracts are available or readily prepared. 另外,通过传统的化学、物理和生物化学方法,容易修饰天然或合成产生的文库和化合物。 Further, through conventional chemical, physical and biochemical means, and a library of modified compounds readily natural or synthetically produced. 可对已知的药理学物质进行定向或随机的化学修饰,例如酰化、烷基化、酯化和/或酰胺化(amidification),以产生结构类似物。 May be made of known pharmacological agents directed or random chemical modifications, such as acylation, alkylation, esterification and / or amidation (amidification), to produce structural analogs.

[0033] 生物活性物质可包括天然生成的蛋白质或天然生成蛋白质的片段。 [0033] The biologically active material may comprise naturally occurring proteins or fragments of naturally occurring proteins. 因此,例如,可以使用包含蛋白质的细胞提取物,或蛋白质细胞提取物的随机或定向消化物。 Thus, for example, you can use random or directed digests of the protein-containing cell extract, a protein or a cell extract. 在此情况下,原核生物和真核生物蛋白质文库可以被制备以便筛选这里所述的体系。 In this case, libraries of prokaryotic and eukaryotic proteins may be prepared so as screening system described herein. 例如可以生成细菌、真菌、病毒和哺乳动物蛋白质的文库,用于筛选目的。 Library may be generated, for example, bacterial, fungal, viral, and mammalian proteins, for screening purposes.

[0034] 生物活性物质可以是约5到约30个氨基酸或约5到约15个氨基酸的肽。 [0034] The biologically active substance may be from about 5 to about 30 amino acids or from about 5 to about 15 amino acid peptide. 肽可以是天然生成蛋白的消化物或随机肽。 Peptides may be digests of naturally occurring proteins or random peptides. 由于一般地,随机肽(或随机核酸)可以被化学合成,因此它们可在任何位置将任何核苷酸或氨基酸整合。 Since Generally, random peptides (or random nucleic acid) can be chemically synthesized, they may be integrated to any nucleotide or amino acid at any position. 可以设计合成过程,以产生随机化的蛋白质或核酸,这使得可以在序列的整个长度范围形成所有或大多数可能的组合,从而形成随机化生物活性物质的文库。 Synthesis process can be designed to generate randomized proteins or nucleic acids, which may be formed such that all or most of the possible combinations over the entire length of the sequence, thus forming a library of randomized bioactive substance.

[0035] 可选地,生物活性物质可以是核酸。 [0035] Alternatively, the biologically active substance may be a nucleic acid. 核酸可以是单链或双链或其混合物。 Nucleic acids can be single or double stranded, or a mixture thereof. 核酸可以是DNA、基因组DNA、cDNA、RNA或杂种,其中核酸包含脱氧核糖核苷酸和核糖核苷酸的任何组合,以及碱基的任何组合,包括尿嘧啶、腺嘌呤、胸腺嘧啶、胞嘧啶、鸟嘌呤、肌苷、黄嘌呤(xanthanine)、次黄嘌呤(hypoxanthanine)、异胞嘧啶、异鸟嘌呤以及碱基对类似物如硝基吡咯和硝基吲哚等。 The nucleic acid may be DNA, genomic DNA, cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine , guanine, inosine, xanthine (xanthanine), hypoxanthine (hypoxanthanine), isocytosine, isoguanine, and base analogs such as nitropyrrole and of nitroindole like.

[0036] 这里公开的条码的应用不限于前述用途,而包括任何涉及靶的检测、鉴定和/或量化的应用。 [0036] The bar code applications disclosed herein are not limited to the aforementioned use, and includes any of detection, identification and / or quantification of a target application relates. 非限定性的应用包括单核苷酸多态性(SNPs)的检测;遗传突变的检测、疾病诊断、法医分析;环境污染和/或病原体的检测;临床诊断测试以及本领域所知的各种其它应用。 Non-limiting applications include detection of single nucleotide polymorphisms (SNPs); detecting environmental pollution and / or pathogen;; detection, diagnosis, forensic analysis of genetic mutations known in the art of clinical diagnostic tests and various other applications.

探针制备寡核苷酸探针 Preparation of probe oligonucleotide probe

[0037] 寡核苷酸的合成方法是本领域熟知的,可以使用任何这样的已知方法。 [0037] The method of synthesizing oligonucleotides are well known in the art, may be used any of such known methods. 例如,可以使用商业可得的寡核苷酸合成仪(如Applied Biosystems,Foster City,CA)制备寡核苷酸。 For example, a commercially available oligonucleotide synthesizer (e.g., Applied Biosystems, Foster City, CA) oligonucleotide was prepared. 可以从商业渠道获得与各种标签连接的核苷酸前体(例如,Molecular Probes,Eugene,OR),并结合进寡核苷酸。 Nucleotide precursor can be obtained (e.g., Molecular Probes, Eugene, OR) connected to the various labels from commercial sources, and incorporated into oligonucleotides. 可选地,可以购买包含各种活性基团的核苷酸前体,例如生物素、地高辛(diogoxigenin)、硫氢基、氨基或羧基。 Alternatively, nucleotide precursors can be purchased containing various reactive groups, such as biotin, digoxigenin (diogoxigenin), sulfhydryl, amino or carboxyl group. 在寡核苷酸合成之后,采用标准的化学方法连接标签。 After oligonucleotide synthesis using standard chemical methods connector tab. 也可以从各种来源得到具有任何期望序列的寡核苷酸,用于标签的连接,其具有或没有活性基团(例如,Miland Certified Reagents,Miland,TX)。 Can be obtained from various sources oligonucleotide has any desired sequence, the label for the connection, with or without reactive groups (e.g., Miland Certified Reagents, Miland, TX). 也可以通过标准酶促方法,制备寡核苷酸探针,例如采用聚合酶链反应(PCRTM)扩增(例如Sambrook等,MolecularCloning:A Laboratory Manual,2nd Ed.,Cold Spring Harbor Press,Cold SpringHarbor,NY,1989;美国专利US 5,279,721;US 4,683,195;US 4,683,202;US4,800,159;US 4,883,750)。 By standard enzymatic methods may also be prepared oligonucleotide probes, for example by polymerase chain reaction (PCRTM) amplification (e.g. Sambrook et al, MolecularCloning:. A Laboratory Manual, 2nd Ed, Cold Spring Harbor Press, Cold SpringHarbor, NY, 1989; US Patent US 5,279,721; US ​​4,683,195; US 4,683,202; US4,800,159; US 4,883,750).

适体探针 Aptamer probe

[0038] 适体是通过被称为SELEX的体外进化过程衍生的寡核苷酸(例如,Brody和Gold,Molecular Biotechnology 74:5-13,2000)。 [0038] aptamer by in vitro evolution process called SELEX derived oligonucleotides (e.g., Brody and Gold, Molecular Biotechnology 74: 5-13,2000). SELEX过程涉及,重复循环暴露潜在的适体(核酸配体)于靶,使得发生结合;从自由核酸配体(freenucleic acid ligand)分离结合配体;扩增结合的配体以及重复结合过程。 SELEX process involves repeated cycles of exposing potential aptamers (nucleic acid ligands) to a target, such binding occurs; from the free nucleic acid ligand (freenucleic acid ligand) separating the ligand binding; binding ligand amplifying and repeating the binding process. 经过许多循环后,可制备出真正针对任何类型生物靶而具有高亲和性及特异性的适体。 After many cycles, it can be prepared for any type of real biological target having high affinity and specificity of aptamers. 由于其小的体积、相对稳定性和制备简便,适体非常适合用作探针。 Because of its small size, relative stability, and simple preparation, aptamers is very suitable for use as probes. 因为适体由寡核苷酸组成,因此它们能容易地结合进核酸型条码中。 Because of aptamer oligonucleotides, so that they can be easily incorporated into nucleic acid type barcode. 制备适体的方法是众所周知的(例如,美国专利US 5,270,163;US 5,567,588;US 5,670,637;US 5,696,249;US 5,843,653)。 The method of preparation of aptamers is well known (e.g., U.S. Patent No. US 5,270,163; US 5,567,588; US 5,670,637; US 5,696,249; US 5,843,653). 可选地,可从商业来源获得针对特定靶标的各种适体(如Somalogic,Boulder,CO)。 Alternatively, commercially available from various sources for a particular target aptamers (e.g. Somalogic, Boulder, CO). 适体是相对较小的分子,其量级为7到50kDa。 Aptamers are relatively small molecules, on the order of 7 to 50kDa.

抗体探针 Antibody probes

[0105] 产生抗体的方法也是本领域熟知的(例如,Harlow和Lane,Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory,Cold SpringHarbor,NY,1988)。 Method [0105] produce antibodies are also known in the art (e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, NY, 1988). 适合用作探针的单克隆抗体也可从许多商业渠道获得。 Monoclonal antibodies suitable for use as probes are also available from many commercial sources. 这些商业抗体可以针对多种靶标起作用。 These commercial antibodies can act against a variety of targets. 使用标准的化学方法,可以将抗体探针与条码结合,如下所述。 Using standard chemical methods, antibody probes may be combined with the bar code, as described below.

[0106] 所公开的方法和组合物并不限于所用探针的类型,并且本领域所知的任何类型探针部分都可以与条码连接并用于所公开的方法中。 [0106] The disclosed methods and compositions are not limited to the type of probe used, and any type of probe moiety known in the art and can be connected to the bar code used in the process disclosed. 这样的探针包括但不限于抗体片段、affibodies、嵌合抗体、单链抗体、配体、结合蛋白、受体、抑制物、底物等。 Such probes include but are not limited to antibody fragments, Affibodies, chimeric antibodies, single chain antibodies, ligands, binding proteins, receptors, inhibitors, substrates and the like.

标签 label

[0101] 在本发明的各种实施方式中,将条码与一个或多个标签连接,以便于检测和/或鉴定。 [0101] In various embodiments of the present invention, one or more bar code tag is attached, to facilitate detection and / or identification. 可以使用本领域已知的任何可检测的标签。 Any detectable label known in the art may be used. 可检测的标签包括但不限于,可以用电学技术、光学技术、分光光度技术、光化学技术、生物化学技术、免疫化学技术或化学技术检测的任何组合物。 Detectable labels include but are not limited to, electricity can techniques, optical techniques, spectrophotometric techniques, photochemical techniques, biochemical techniques, or immunochemical techniques to detect any chemical composition. 标签包括但不限于传导的、发光的、荧光的、化学发光的、生物发光的和发磷光的部分;量子点;纳米颗粒;金属纳米颗粒;金纳米颗粒;银纳米颗粒;色原体;抗体;抗体片段;基因工程抗体;酶;底物;辅因子;抑制物;结合蛋白;磁性颗粒以及自旋标记化合物。 Tag including but not limited to conducting, luminescent, fluorescent, chemiluminescent, bioluminescent and phosphorescent moiety; quantum dot; nanoparticles; metal nanoparticles; gold particles; silver nanoparticles; chromogen; antibody ; antibody fragment; engineered antibody; an enzyme; a substrate; cofactors; inhibitors; binding protein; magnetic particles and spin label compounds. (美国专利US 3,817,837;US 3,850,752;US 3,939,350;US 3,996,345;US 4,277,437;US 4,275,149和US 4,366,241。)拉曼标签 (US Patent US 3,817,837; US 3,850,752; US 3,939,350; US 3,996,345; US 4,277,437; US 4,275,149 and US 4,366,241.) Raman label

[0102] 使用的拉曼标签的非限定性例子包括TRIT(四甲基罗丹明异硫醇)、NBD(7-硝基苯-2-氧杂-1,3-二唑)、德克萨斯红染料、邻苯二甲酸、对苯二酸、间苯二酸、甲酚固紫、甲酚蓝紫、亮甲酚蓝、对氨基苯甲酸、藻红、生物素、地高辛、5-羧基-4′,5′-二氯-2′,7′-二甲氧基荧光素、TET(6-羧基-2′,4,7,7′-四氯荧光素)、HEX(6-羧基-2′,4,4′,5′,7,7′-六氯荧光素)、Joe(6-羧基-4′,5′-二氯-2′,7′-二甲氧基荧光素)、5-羧基-2′,4′,5′,7′-四氯荧光素、5-羧基荧光素、5-羧基罗丹明、Tamra(四甲基罗丹明)、6-羧基罗丹明、Rox(羧基-X-罗丹明)、R6G(罗丹明6G)、酞菁、偶氮次甲(azomethine)、花菁(如Cy3、Cy3.5、Cy5)、黄嘌呤、琥珀酰荧光素、N,N-二乙基-4-(5′-偶氮苯三唑基)-苯胺以及氨基吖啶。 [0102] Non-limiting examples of Raman labels used include TRIT (tetramethyl rhodamine iso-thiol), NBD (7- nitrobenz-2-oxa-1,3-diazole), Texas Adams red dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5 - carboxy-4 ', 5'-dichloro-2', 7'-dimethoxy fluorescein, TET (6- carboxy-2 ', 4,7,7'-tetrachlorofluorescein), HEX (6 - carboxy-2 ', 4,4', 5 ', 7,7'-hexachlorofluorescein), Joe (6- carboxy-4', 5'-dichloro-2 ', 7'-dimethoxy fluorescein), 5-carboxy-2 ', 4', 5 ', 7'-tetrachloro-fluorescein, 5-carboxyfluorescein, 5-carboxy rhodamine, Tamra (tetramethylrhodamine), 6-carboxy Rodin Ming, Rox (carboxy -X- rhodamine), R6G (rhodamine. 6G), phthalocyanines, azo methine (Azomethine), cyanine (e.g., Cy3, Cy3.5, Cy5), xanthines, succinyl fluorescein , N, N- diethyl-4- (5'-triazolyl azobenzene) - aniline, and amino acridine. 这些以及其它拉曼标签可以从商业渠道获得(例如,Molecular Probes,Eugene,OR)。 These and other Raman labels may be obtained (e.g., Molecular Probes, Eugene, OR) are commercially available.

[0103] 多环芳香化合物一般可用作拉曼标签。 [0103] Usually polycyclic aromatic compounds as Raman labels. 可用的其它标签包括氰化物、硫醇、氯、溴、甲基、磷和硫。 Other labels may be used include cyanide, thiol, chlorine, bromine, methyl, phosphorus and sulfur. 在某些实施方式中,碳纳米管可用作拉曼标签。 In certain embodiments, the carbon nanotubes can be used as Raman tags. 标签在拉曼光谱中的用法是已知的(例如美国专利US 5,306,403和US 6,174,677)。 Use labels in Raman spectroscopy is known (e.g., U.S. Patent No. US 5,306,403 and US 6,174,677).

[0104] 拉曼标签可以直接与条码连接或者通过各种接头化合物与之连接。 [0104] Raman labels and bar code can be directly connected or connected thereto via various linker compounds. 与拉曼标签共价连接的核苷酸可以从标准商业来源得到(例如,Roche MolecularBiochemicals,Indianapolis,IN;Promega Corp.,Madison,WI;Ambion,Inc.,Austin,TX;Amersham Pharmacia Biotech,Piscataway,NJ)。 Raman labels covalently connected nucleotides can be obtained from standard commercial sources (e.g., Roche MolecularBiochemicals, Indianapolis, IN; Promega Corp., Madison, WI; Ambion, Inc., Austin, TX; Amersham Pharmacia Biotech, Piscataway, NJ). 含有活性基团、设计与其它分子如核苷酸或氨基酸进行共价反应的拉曼标签是商业可得的(例如,Molecular Probes,Eugene,OR)。 Raman labels contain reactive groups designed for covalent reaction with other molecules such as nucleotides or amino acids are commercially available (e.g., Molecular Probes, Eugene, OR).

荧光标签 Fluorescent labels

[0105] 可能使用的荧光标签包括但不限于荧光素、5-羧基荧光素(FAM)、2′7′-二甲氧基-4′5′-二氯-6-羧基荧光素(JOE)、罗丹明、6-羧基罗丹明(R6G)、N,N,N′,N′-四甲基-6-羧基罗丹明(TAMRA)、6-羧基-X-罗丹明(ROX)、4-(4′-二甲基氨基苯基偶氮)苯甲酸(DABCYL)以及5-(2′-氨乙基)氨基萘-1-磺酸(EDANS)。 [0105] fluorescent labels may be used include but are not limited to, fluorescein, 5-carboxyfluorescein (FAM), 2'7'- -4'5'- dimethoxy-dichloro-6-carboxyfluorescein (JOE) , rhodamine, 6-carboxyrhodamine (R6G), N, N, N ', N'- tetramethyl-6-carboxy rhodamine (TAMRA), 6-carboxy -X- rhodamine (ROX), 4- (4'-dimethylaminophenylazo) benzoic acid (, DABCYL), and 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS). 其它可用的荧光标签在本领域是已知的(例如,美国专利US 5,866,336)。 Other useful fluorescent labels are known (e.g., U.S. Patent No. US 5,866,336) in the art. 各种荧光标签都可从商业渠道获得,例如Molecular Probes(Eugene,OR)。 Various fluorescent tags are available from commercial sources, such as Molecular Probes (Eugene, OR). 标记分子的荧光检测方法也是本领域熟知的,并且可以使用任何这样的已知方法。 Fluorescence detection method labeled molecules are also known in the art, and may utilize any such known methods.

[0106] 使用的发光标签包括但不限于稀土金属穴状化合物、三双吡啶二胺铕(europium trisbipyridine diamine)、铕穴状化合物或螯合物、三双吡啶铽(Tbtrisbipyridine)、二胺、二花青苷、La Jolla蓝染料、别藻蓝蛋白(allopycocyanin)、别花青素B(allococyanin B)、藻青素C、藻青素R、硫胺素、藻胆青素(phycoerythrocyanin)、藻红素R、上转换或下转换磷(up-converting ordown-converting phosphor)、萤光素或吖啶酯(acridinium ester)。 [0106] The use of luminescent labels include but are not limited to rare-earth metal cryptate, tris bipyridine diamine europium (europium trisbipyridine diamine), europium cryptate or chelate, tris bipyridyl terbium (Tbtrisbipyridine), diamine, anthocyanin, La Jolla blue dye, allophycocyanin (allopycocyanin), not anthocyanins B (allococyanin B), phycocyanin C, phycocyanin R, thiamine, phycobiliproteins astaxanthin (phycoerythrocyanin), sp P conversion (up-converting ordown-converting phosphor) under red pigment R, or the conversion, luciferin, or acridinium esters (acridinium ester).

纳米颗粒标签 Nanoparticle labels

[0107] 纳米颗粒可用作标签,例如在用各种方法检测条码时。 [0107] Nanoparticles may be used as labels, for example, when the bar code is detected by various methods. 制备纳米颗粒的方法是已知的(例如美国专利US 6,054,495;US 6,127,120;US 6,149,868;Lee和Meisel,J.Phys.Chem.86:3391-3395,1982)。 The method of preparing nanoparticles are known (e.g. U.S. Patent No. US 6,054,495; US 6,127,120; US 6,149,868; Lee and Meisel, J.Phys.Chem.86: 3391-3395,1982). 纳米颗粒也可以从商业途径得到(例如,Nanoprobes Inc.,Yaphank,NY;Polysciences,Inc.,Warrington,PA)。 Nanoparticles may also be obtained (e.g., Nanoprobes Inc., Yaphank, NY; Polysciences, Inc., Warrington, PA) from commercially. 虽然金或银纳米颗粒通常被用作标签,但纳米颗粒的任何类型或组合物都可以与条码连接,用作标签。 Although gold or silver nanoparticles are commonly used as a label, but any type or composition of the nanoparticles can be connected to the bar code, used as a label.

[0108] 使用的纳米颗粒可以是纳米颗粒的无规则聚集体(胶状纳米颗粒)。 [0108] The use of nanoparticles may be random aggregates of nanoparticles body (colloidal nanoparticles). 可选地,纳米颗粒可以被交联,以产生特殊的纳米颗粒聚集体,例如二聚体、三聚体、四聚体或其它聚集体。 Alternatively, nanoparticles may be cross-linked to produce particular aggregates of nanoparticles, such as dimers, trimers, tetramers or other aggregates. 包含选择数目纳米颗粒的聚集体(二聚体、三聚体等)可以用已知的方法进行富集或提纯,例如在蔗糖溶液中的超离心法。 An aggregate that comprises a selected number of nanoparticles (dimers, trimers, etc.) may be enriched or purified, e.g. ultracentrifugation in sucrose solution using known methods.

[0109] 适合用于连接条码的修饰纳米颗粒是商业可得的,例如Nanoprobes,Inc.(Yaphank,NY)的Nanogold纳米颗粒。 [0109] Modified nanoparticles suitable for connecting the barcode are commercially available, e.g. Nanoprobes, Inc. (Yaphank, NY) Nanogold the nanoparticles. Nanogold纳米颗粒可以用单个或多个马来酰亚胺、胺或其它基团连接在每个纳米颗粒上而获得。 Nanogold nanoparticles may be single or multiple maleimide, amine or other groups attached per nanoparticle was obtained. 使用各种已知的接头化合物,可将这种修饰的纳米颗粒连接于条码。 Using a variety of known linker compounds, such modifications can be coupled to the bar code nanoparticles.

金属标签 Metal tag

[0110] 标签可包括亚微米大小的金属标签(例如,Nicewarner-Pena等,Science 294:137-141,2001)。 [0110] tag may include submicron-sized metal tag (e.g., Nicewarner-Pena et, Science 294: 137-141,2001). Nicewamer-Pena等(2001)公开了编码亚微米条纹(stripe)的多金属微棒(microrod)的方法,其由不同类型的金属组成。 Nicewamer-Pena et al. (2001) discloses a method for encoding submicron stripes (stripe) multimetal microrods (microrod), consisting of different types of metal. 该系统可以产生大量可区分的标签-用两种金属可达4160个,用三种不同金属可多达8×105个。 The system may generate large number of distinguishable labels - up to 4160 using two metals, up to three different metal th 8 × 105. 这样的标签可与条码连接并被检测。 Such tags may be attached to barcodes and detected. 将金属颗粒如金或银与寡核苷酸和其它类型分子连接的方法是本领域已知的(例如美国专利US 5,472,881)。 Metal particles such as silver or gold method with oligonucleotides and other types of molecules are known in the art (e.g. U.S. Patent No. US 5,472,881).

富勒烯标签 Fullerenes label

[0111] 富勒烯也可用作条码标签。 [0111] Fullerene barcode labels may also be used. 产生富勒烯的方法是已知的(例如美国专利US 6,358,372)。 Method of producing fullerenes are known (e.g. U.S. Patent No. US 6,358,372). 通过类似于下面所述的关于碳纳米管的方法,可以对富勒烯进行衍生并连接于其它分子。 It can be derivatized by the methods described below with respect to fullerenes and carbon nanotubes connected to the analogy to other molecules. 标记富勒烯的条码可以用例如各种技术进行鉴定。 Labeled fullerenes barcodes can be identified by a variety of techniques, for example.

[0112] 可连接于条码并可检测的其它类型的已知标签被考虑在内。 Other types of [0112] may be connected to the detection and bar code labels known are contemplated. 可能使用的标签的非限定性例子包括量子点(例如,Schoenfeld等,Proc.7th Int.Conf.onModulated Semiconductor Structures,Madrid,pp.605-608,1995;Zhao等,1st Int.Conf.on Low Dimensional Structures and Device,Singapore,pp.467-471,1995)。 Non-limiting examples of labels may be used include quantum dots (e.g., Schoenfeld, etc., Proc.7th Int.Conf.onModulated Semiconductor Structures, Madrid, pp.605-608,1995; Zhao et, 1st Int.Conf.on Low Dimensional Structures and Device, Singapore, pp.467-471,1995). 量子点和其它类型的标签也可以从商业渠道获得(例如,Quantum Dot Corp.,Hayward,CA)。 Quantum dots and other types of labels can also be obtained (e.g., Quantum Dot Corp., Hayward, CA) are commercially available.

碳纳米管标签 Carbon nanotubes label

[0113] 碳纳米管如单壁碳纳米管(SWNTs)也可用作标签。 [0113] The carbon nanotubes single wall carbon nanotubes (SWNTs) may be used as the label. 纳米管可以用例如拉曼光谱进行检测(例如,Freitag等,Phys.Rev.B 62:R2307-R2310,2000)。 Nanotubes can be detected by, for example, Raman spectroscopy (e.g., Freitag, etc., Phys.Rev.B 62: R2307-R2310,2000). 碳纳米管的特性,如电或光性质,至少部分取决于纳米管的大小。 Properties of carbon nanotubes, electrical or optical properties, at least in part on the size of the nanotube.

[0114] 可以用本领域已知的各种技术制造碳纳米管,包括但不限于碳电弧放电法、通过烃催化裂解的化学气相沉积法、等离子体辅助的化学气相沉积法、催化含金属石墨靶的激光烧蚀或浓缩相电解(condensed-phase electrolysis)。 [0114] can be used a variety of techniques known in the art for producing carbon nanotubes, including but not limited to, carbon arc discharge method, catalytic cracking of hydrocarbons by chemical vapor deposition, plasma assisted chemical vapor deposition method, a catalytic metal-containing graphite laser ablation target phase electrolysis or concentrated (condensed-phase electrolysis). (参见,例如,美国专利US 6,258,401;US 6,283,812和US 6,297,592)。 (See, e.g., U.S. Patent No. US 6,258,401; US ​​6,283,812 and US 6,297,592). 使用本领域已知的任何方法,根据纳米管长度和直径,可将包括不同长度碳纳米管混合物的组合物分离成不连续的大小级别。 Using any method known in the art, according to the length and diameter of the nanotube, a carbon nanotube composition may comprise a mixture of different lengths separated into discrete size level. 例如,可以用质谱法,对纳米管进行大小分类(参见,Parker等,“High yield synthesis,separation and mass spectrometriccharacterization of fullerene C60-C266,”J.Am.Chem.Soc.113:7499-7503,1991)。 For example, mass spectrometry may be used, size classification of the nanotubes (see, Parker et, "High yield synthesis, separation and mass spectrometriccharacterization of fullerene C60-C266," J.Am.Chem.Soc.113: 7499-7503,1991 ). 碳纳米管也可以从商业渠道得到,例如CarboLex(Lexington,KY)、NanoLab(Watertown,MA)、Materials and Electrochemical Research(Tucson,AZ)或CarbonNano Technologies Inc.(Houston,TX)。 Carbon nanotubes can also be obtained from commercial sources, such as CarboLex (Lexington, KY), NanoLab (Watertown, MA), Materials and Electrochemical Research (Tucson, AZ) or CarbonNano Technologies Inc. (Houston, TX).

[0115] 可以用活性基团对碳纳米管进行衍生,以便于附着条码。 [0115] reactive groups can be derivatized carbon nanotubes, so that the bar code attached. 例如,纳米管可以被衍生为含有羧酸基团(美国专利US 6,187,823),羧酸基团可以用碳二亚胺交联剂与胺连接。 For example, nanotubes may be derivatized to contain carboxylic acid groups (U.S. Patent No. US 6,187,823), a carboxylic acid group may be attached with a carbodiimide crosslinking agent and an amine.

核苷酸标签 Nucleotide tag

[0116] 核苷酸或碱基,例如腺嘌呤、鸟嘌呤、胞嘧啶或胸腺嘧啶可以用于标记除寡核苷酸和核酸以外的分子条码。 [0116] nucleotides or bases, such as adenine, guanine, cytosine or thymine can be used to label oligonucleotides and other molecules other than a nucleic acid barcode. 例如,基于肽的分子条码可以用核苷酸或嘌呤或嘧啶碱基标记。 For example, peptide-based molecules can be labeled with a bar code or a nucleotide purine or pyrimidine base. 其它类型的嘌呤或嘧啶或其类似物也可用作标签,例如尿嘧啶、肌苷、2,6-二氨基嘌呤、5-氟-脱氧胞嘧啶、7-脱氮-脱氧腺嘌呤或7-脱氮-脱氧鸟嘌呤。 Other types of purine or pyrimidine or an analog thereof can also be used as a label, e.g. uracil, inosine, 2,6-diaminopurine, 5-fluoro - deoxycytidine, 7-deaza - deoxyadenosine or 7 denitrification - deoxyguanosine. 其它标签包括碱基类似物。 Other labels include base analogs. 碱基是没有糖类或磷酸的含氮环结构。 No nucleotide sugars or phosphoric acid is a nitrogen-containing ring structure. 这些标签可以用光学技术如拉曼或荧光光谱进行检测。 These tags can be by optical techniques such as Raman spectroscopy or fluorescence detection. 当要检测的靶分子是核酸或寡核苷酸时,使用核苷酸或核苷酸类似物标签是不合适的,这是因为条码的标签部分可能会与不同的靶分子杂交,而不与探针部分杂交。 When the target molecule to be detected is a nucleic acid or an oligonucleotide, nucleotide or nucleotide analog label used is inappropriate, since the bar code tag portion may hybridize with different target molecules, rather than with probe hybridizes.

氨基酸标签 Amino acid tag

[0117] 氨基酸也可用作标签。 [0117] amino acids may also be used as a label. 可能用作标签的氨基酸包括当不限于苯丙氨酸、酪氨酸、色氨酸、组氨酸、精氨酸、半胱氨酸和蛋氨酸。 Amino acid used as a label may include, if not limited to, phenylalanine, tyrosine, tryptophan, histidine, arginine, cysteine ​​and methionine.

交联剂 Crosslinkers

[0118] 双功能交联剂可用于各种目的,例如将标签与条码连接。 [0118] Bifunctional cross-linking agent may be used for various purposes, for example, connected to the bar code label. 双功能交联剂可以根据其功能基如氨基、胍基、吲哚或羧基特定基团的专一性进行划分。 Cross-linkers can be classified according to their specific functional group such as amino, guanidino, indole, or carboxyl specific groups will be. 在这些交联剂中,涉及自由氨基的交联剂由于其商业可得性、易于合成以及应用时温和的反应条件而受青睐(美国专利US 5,603,872和US 5,401,511)。 Among these crosslinking agents, crosslinking agents because it relates to a free amino commercial availability, ease of synthesis and the mild reaction conditions applied when favored (U.S. Patent No. US 5,603,872 and US 5,401,511). 可能使用的交联剂包括戊二醛(GAD)、双功能环氧乙烷(OXR)、乙二醇二缩水甘油醚(EGDE)和碳二亚胺,例如1-乙基-3-(-二甲基氨基丙基)碳二亚胺(EDC)。 Crosslinking agents that may be used include glutaraldehyde (of GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and carbodiimides, such as 1-ethyl-3 - (- dimethylaminopropyl) carbodiimide (EDC).

条码检测 Barcode detection

[0119] 可以用本领域已知的任何方法检测条码。 [0119] Barcodes can be detected by any method known in the art. 例如,可用荧光光谱法检测条码。 For example, barcode detection of fluorescence spectroscopy can be used. 可将几个荧光染料连接在单个条码上。 Several fluorescent dye can be connected on a single bar code. 条码中染料的量和化学性质将决定条码的荧光发射图谱。 Barcode dyes and chemical properties will determine the amount of fluorescence emission spectra barcode. 对于给定的条码组合物而言,信号可受到标签之间的相对距离影响,这是因为可能存在共振能量的转移。 For a given barcode compositions, the impact signal may be a relative distance between the tags, due to the presence of possible resonance energy transfer.

[0120] 在其他实施方式中,可用拉曼光谱法检测条码。 [0120] In other embodiments, the barcode detection Raman spectroscopy can be used. 可将各种拉曼标签与条码连接,以便用已知的拉曼光谱技术进行检测,例如SERS(表面增强拉曼光谱法)。 May be a variety of Raman labels and bar codes are connected, for detecting Raman spectroscopy using known techniques, for example, the SERS (surface enhanced Raman spectroscopy). 除了连接的拉曼标签外,条码骨架本身也可用作拉曼标签。 In addition to the Raman label attached barcode backbone itself as Raman labels. DNA分子的不同碱基组合物产生不同的拉曼信号,这些信号可用于鉴定基于DNA的条码。 Different base composition of the DNA molecules to produce different Raman signals, these signals may be used to identify DNA-based bar code. 各种具体的检测模式将在下面讨论。 Various specific mode of detection will be discussed below.

拉曼光谱法用于拉曼光谱法的表面 Raman Spectroscopy Raman spectroscopy surface

[0121] 各种形式的拉曼光谱法都利用标记(条码)分子接近表面所引起的拉曼信号的增强。 [0121] Various forms of Raman spectroscopy utilize labeled (bar code) molecules adjacent the surface enhanced Raman signals caused. 在某些方法中,例如表面增强拉曼光谱法(SERS)或表面增强共振拉曼光谱法(SERRS)中,与拉曼活性金属表面如金、银、铝、铂、铜或其他金属的接近可增强拉曼信号达6或7个数量级。 In some methods, such as surface enhanced Raman spectroscopy (SERS) or surface enhanced resonance Raman spectroscopy (SERRS), as close to the metal surface and the Raman-active gold, silver, aluminum, platinum, copper or other metals enhanced Raman signal can be up to 6 or 7 orders of magnitude.

其它类型的化合物也可用于增强SERS中的信号,例如LiF、NaF、KF、LiCl、NaCl、KCl、LiBr、NaBr、KBr、LiI、NaI和KI。 Other types of compounds may also be used to enhance the SERS signal, e.g. LiF, NaF, KF, LiCl, NaCl, KCl, LiBr, NaBr, KBr, LiI, NaI and KI. 具体地,LiCl已被证明能提高具体分析物(如dAMP、脱氧腺苷、腺苷、和腺嘌呤)的相对信号强度2到100倍。 In particular, LiCl has been shown to increase the specific analyte (e.g. dAMP, deoxyadenosine, adenosine and adenine) relative signal strength from 2 to 100 times. 取决于感兴趣的分析物,与通常使用的NaCl比较,LiCl提高相对强度2倍以上。 Depending on the analyte of interest, compared to the commonly used NaCl, LiCl enhance the relative intensity of more than two times. 在其他实施方式中,对于分析物如脱氧鸟苷-一磷酸(deoxyguanosine-monophosphate)(dGMP),NaBr或NaI比LiCl好。 In other embodiments, the analytes, such as for deoxyguanosine - monophosphate (deoxyguanosine-monophosphate) (dGMP), NaBr or NaI better than LiCl.

拉曼检测器 Raman detector

[0122] 各种拉曼检测的方法在本领域是已知的。 [0122] Various methods of Raman detection is known in the art. 美国专利US 6,002,471公开了可用的拉曼检测装置的一个例子。 U.S. Patent No. US 6,002,471 discloses an example of a Raman detection means available. 正如所公开的,激发光束由532nm波长的Nd:YAG激光器或365nm波长的Ti:蓝宝石激光器产生。 As disclosed, the light beam from the Nd 532nm excitation wavelength: YAG laser wavelength of 365nm or a Ti: sapphire laser generates. 可使用脉冲激光光束或连续激光光束。 Using pulsed laser light or a continuous laser beam. 激发光束通过共焦光学系统和显微镜物镜,并被聚焦在靶区域上。 Excitation beam passes through confocal optics and a microscope objective, and is focused on the target area. 来自拉曼标记的拉曼发射光聚集于显微镜物镜和共焦光学系统并连到单色仪(monochromonator),以分离光谱。 The Raman emission light from the Raman label accumulated in the microscope objective and the confocal optics and is connected to the monochromator (monochromonator), to separate the spectrum. 共焦光学系统包括分色滤光器、阻挡层滤波器、共焦小孔、透镜和平面镜的组合,以减小背景信号。 Confocal optical system comprises a dichroic filter, a barrier layer filter, the confocal aperture, combination of lenses and mirrors to reduce background signal. 和共焦光学系统一样,也可使用标准的全视场光学系统。 And a confocal optical systems, also use a standard full field optical system. 用任何已知的拉曼检测器检测信号。 Any known Raman detection signals.

[0123] 检测装置的可选例子在例如美国专利US 5,306,403中公开,其包括Spex Model 1403型双光栅分光光度计,它装备有镓-砷(GaAs)光电倍增管(RCAModel C31034或Burle Industries Model C3103402),以单光子计数模式进行操作。 [0123] Alternatively, for example, in an example of the detection apparatus disclosed in U.S. Patent No. US 5,306,403, which comprises a Spex Model 1403 double-grating spectrophotometer equipped with a gallium it - arsenic (GaAs) photomultiplier tube (RCAModel C31034 or Burle Industries Model C3103402 ), operated in a single photon counting mode.

[0124] 另一个示例性拉曼检测装置包括激光和拉曼检测器。 [0124] Another example of a Raman detection means comprises a laser and a Raman detector. 激发光束由近红外波长(750~950nm)的钛:蓝宝石激光器(SpectraPhysics公司的Tsunami)或者785nm或830nm的镓铝砷二极管激光器(Process Instruments公司的PI-ECL系列)产生。 Titanium excitation beam from the near-infrared wavelength (750 ~ 950nm) of: sapphire laser (Tsunami SpectraPhysics's) of 830nm or 785nm or gallium aluminum arsenide diode laser (Process Instruments Company PI-ECL series) is generated. 可使用脉冲激光束或连续激光束。 Pulsed laser beams or may be a continuous laser beam. 激发光束由分色镜(Kaiser Optical公司的全息陷波滤波器或者Chroma或Omega Optical公司的干扰滤波器)反射为具有聚集光束的共线几何关系(collinear geometry)。 Excitation beam by the dichroic mirror (Kaiser Optical holographic notch filter's or company's Chroma or Omega Optical interference filters) reflecting a collinear geometry with (collinear geometry) light beams are collected. 反射的光束通过显微镜物镜(NikonLU系列),被聚焦在条码结合的靶标所在的区域上。 The light beam reflected by the microscope objective (NikonLU series), the bar code is focused on an area where the binding target. 拉曼散射光由同一显微镜物镜聚集,并经过分色镜到达拉曼检测器。 Raman scattered light is collected by the same microscope objective through the dichroic mirror and the detector to Dalaman. 拉曼检测器包括聚焦透镜、摄谱仪和阵列式检测器。 Raman detector comprises a focusing lens, a spectrograph, and an array detector. 聚焦透镜将拉曼散射光聚焦,通过摄谱仪的入口。 The focusing lens focused the Raman scattered light through the entrance of the spectrograph. 摄谱仪(RoperScientific)包括光珊,其按波长使光分散。 Spectrograph (RoperScientific) comprises a light coral, which disperse light by wavelength. 分散的光成像在阵列检测仪上(RoperScientific公司的背景照明深度耗尽CCD照相机)。 Dispersed light is imaged on the detector array (RoperScientific's background illumination deeply depleted CCD camera). 阵列检测器与控制器电路相连,并与计算机连接,以传输数据及控制检测器功能。 Array detector circuit coupled to the controller, and a computer connected to the detector control and data transfer functions.

[0125] 可选的激发光源包括氮激光器(Laser Science Inc.)和氦-镉激光器(Liconox)(美国专利US 6,174,677)。 [0125] Alternatively the excitation light source comprises a nitrogen laser (Laser Science Inc.) and helium - cadmium laser (Liconox) (U.S. Patent No. US 6,174,677). 激发光束用带通滤波器(Corion)进行光谱纯化并用6X物镜(Newport,Model L6X)进行聚焦。 Excitation beam spectrally purified with a bandpass filter (Corion) and 6X focusing lens (Newport, Model L6X). 物镜用于激发感兴趣的分子和聚集拉曼信号(Kaiser Optical Systems,Inc.,Model HB 647-26N18)。 An objective lens for gathering and excited molecules of the Raman signal of interest (Kaiser Optical Systems, Inc., Model HB 647-26N18). 全息陷波滤波器(Kaiser Optical Systems,Inc.)用于减小Rayleigh发散辐射。 Holographic notch filter (Kaiser Optical Systems, Inc.) Used to reduce Rayleigh diverging radiation. 可使用其他类型的检测器,例如带电注入器件(charged injection device)、光电二极管阵列或光电晶体管阵列。 Other types of detectors may be used, such as charged injection devices (charged injection device), photodiode arrays or phototransistor arrays.

[0126] 对于多元条码,可选的检测系统包括译码重叠条码的差异。 [0126] For the polyhydric barcode detection system includes an optional difference overlapping barcode decoding. 区分这些条码的一个方法可以是标准的DSP(数字信号处理)方法,使得例如可以区分信号单元中不同条码元素之间的距离(激发引起的波长吸收或位移、物理距离、隧道效应传导率等)。 A method of distinguishing the bar code may be a standard DSP (digital signal processing) methods, for example, can be distinguished so that the distance between the signal units in different barcode elements (excitation wavelength absorption caused or displacement, distance and the like tunneling conductivity) .

[0127] 可以使用本领域已知的任何合适形式或构造的拉曼光谱法或相关技术,例如常规拉曼散射、共振拉曼散射、SERS、表面增强共振拉曼散射、相干反斯托克斯拉曼光谱法(CARS)、受激拉曼散射、逆转拉曼光谱法、受激增益拉曼光谱法、超拉曼散射、分子光学激光检查器(MOLE)或拉曼微探针或拉曼显微镜法或共焦拉曼显微分光光度计法、三维或扫描拉曼、拉曼饱和光谱法、时间分辨共振拉曼、拉曼去耦光谱法或UV-拉曼显微镜法。 [0127] known in the art may be used in any suitable form or configuration of Raman spectroscopy or related techniques, such as normal Raman scattering, resonance Raman scattering, the SERS, surface enhanced resonance Raman scattering, coherent anti-Stokes Raman spectroscopy (the CARS), stimulated Raman scattering, Raman spectroscopy reversed, stimulated gain Raman spectroscopy, ultra-Raman scattering, molecular optical laser checker (MOLE) or Raman microprobe or Raman Raman microscopy or confocal microscopy spectrophotometer, the three-dimensional or scanning Raman, Raman saturation spectroscopy, time resolved resonance Raman, Raman decoupling spectroscopy or UV- Raman microscopy.

微-电-机械系统(Micro-Electro-Mechanical Systems)(MEMS) The micro - electric - mechanical system (Micro-Electro-Mechanical Systems) (MEMS)

[0128] 可将条码制备、使用和/或检测的设备与较大设备和/或系统相结合。 [0128] barcode can be prepared, used and / or detection device with a larger device and / or combined systems. 在某些实施方式中,这些设备包括微-电-机械系统(MEMS)。 In certain embodiments, the devices include a micro - electric - mechanical systems (MEMS). MEMS是包括机械元件、传感器、执行元件(actuator)和电子元件(electronics)的集成系统。 MEMS is a mechanical elements, sensors, actuators (Actuator) and electronic components (Electronics) integrated system. 所有这些组件都可以在硅基或等同衬底的常规芯片上通过微加工技术制造(例如,Voldman等,Ann.Rev.Biomed.Eng.1:401-425,1999)。 All of these components can be on a silicon chip or equivalent substrate by conventional techniques for producing micro-machining (e.g., Voldman the like, Ann.Rev.Biomed.Eng.1: 401-425,1999). MEMS的传感器组件用于测量机械、热、生物、化学、光学和/或磁现象,以便检测条码。 MEMS sensor assembly for measuring mechanical, thermal, biological, chemical, optical and / or magnetic phenomena to detect the bar code. 电子元件处理来自传感器的信息,并控制执行组件,例如泵、阀、加热器等,从而控制MEMS的功能。 Processing the electronic component information from the sensors, and controls the execution components, such as pumps, valves, heaters, etc. thereby controlling the function of the MEMS.

[0129] MEMS的电子组件可使用集成电路(IC)工艺(CMOS或两极处理工艺(Bipolar process))制造。 [0129] MEMS electronic components using an integrated circuit (IC) process (CMOS or bipolar process technology (Bipolar process)) manufactured. 这些电子组件可使用制造计算机芯片的照相平版印刷法和蚀刻法而带有图案。 These electronic components may be patterned using photolithography and etching method of manufacturing computer chips. 微机械组件用兼容的“显微机械加工”方法进行制造,其选择性地蚀刻硅芯片部分或者加入新的结构层,以形成机械和/或机电组件。 The micromechanical components with compatible "micromachining" methods for producing, selectively etch silicon chip portion or add new structural layers to form the mechanical and / or electromechanical components.

[0130] MEMS制造中的基本技术包括:在衬底上沉积薄膜层物质;用平版印刷法将有图案的掩模施用在膜的顶部;以及选择性地时刻膜。 Basic Technique [0130] MEMS manufacture comprising: depositing a thin layer of material on a substrate; a planographic printing method using the mask pattern is applied with a top of the films; films and selectively time. 薄膜可以是几纳米到100微米范围。 The film may be a few nanometers to 100 micrometers. 可用的沉积技术包括化学方法如化学气相沉积(CVD)、电镀、晶体取向附生(epitaxy)和热氧化以及物理方法如物理气相沉积(PVD)和浇铸。 Available deposition techniques include chemical methods such as chemical vapor deposition (CVD), plating, crystal epitaxy (Epitaxy), and thermal oxidation and physical methods such as physical vapor deposition (PVD) and casting. 也可使用制造纳米机电系统的方法(参见,例如,Craighead,Science 290:1532-36,2000)。 It may also be used in methods for making nano electromechanical systems (see, e.g., Craighead, Science 290: 1532-36,2000).

[0131] 在一些实施方式中,设备和/或检测器可与各种充满流体的室相连,例如微流体通道(microfluidic channel)或纳米通道(nanochannel)。 [0131] In some embodiments, the devices and / or detector may be connected to the chamber filled with a variety of fluids, for example microfluidic channels (microfluidic channel) or nanochannel (nanochannel). 这些以及其他设备组件可以形成单个元件,例如以芯片(如半导体芯片)和/或微毛细管或微流体芯片(microfluidic chip)的形式。 These and other device components may form a single element, for example (e.g. semiconductor chips) and / or microcapillary or microfluidic chips in a chip form (microfluidic chip) is. 可选地,各个组件可以分开制造,再组合在一起。 Optionally, individual components can be manufactured separately, then combined. 在这些芯片中使用的任何已知的物质可用于所公开的设备,例如硅、氧化硅、聚二甲基硅氧烷(PDMS)、聚甲基丙烯酸甲酯(PMMA)、塑料、玻璃、石英等。 Any known materials used in these chips can be used in the apparatus disclosed in, for example, silicon, silicon oxide, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), plastic, glass, quartz Wait.

[0132] 批量制造芯片的技术在计算机芯片制造和/或微毛细管芯片制造中是众所周知的。 [0132] batch fabrication technique chip computer chip manufacture and / or microcapillary chip manufacture are well known. 这些芯片可以用本领域已知的任何方法制造,例如光蚀刻和蚀刻、激光烧蚀、注塑成形、浇铸、分子束外延、蘸水笔式纳米光刻法、化学气相沉积(CVD)制造、电子光束或聚焦离子束技术或压印技术。 These chips may be manufactured by any method known in the art, for example, photo-etching and etching, laser ablation, injection molding, casting, molecular beam epitaxy, dip-pen nanolithography, chemical vapor deposition (CVD) manufacturing, electronic beam or focused ion beam technology or imprinting techniques. 非限定性例子包括二氧化硅的传统铸型、干蚀刻;以及电子束平板光刻法。 Non-limiting examples of the mold include conventional silica, dry etching; plate and an electron beam lithography. 制造纳米机电系统的方法可用于某些实施方式。 The method of manufacturing a nanoelectromechanical systems may be used in certain embodiments. (参见,例如,Craighead,Science 290:1532-36,2000。)各种形式的微制造芯片是商业可得的,例如从Caliper Technologies Inc.(MountainView,CA)和ACLARA BioSciences Inc.(Mountain View,CA)获得。 (See, e.g., Craighead, Science 290:. 1532-36,2000) Various forms of microfabricated chips are commercially available, for example from Caliper Technologies Inc. (MountainView, CA) and ACLARA BioSciences Inc. (Mountain View, CA).

[0133] 在某些实施方式中,可选择部分或所有的设备对于激发和发射频率的电磁辐射是透明的,以用于例如拉曼光谱法所进行的条码检测。 [0133] In certain embodiments, some or all of the selectable devices is transparent to the excitation and emission frequencies of electromagnetic radiation, for example, bar code detector Raman spectroscopy performed. 合适的组件可以由物质如玻璃、硅、石英或其它任何光学透明物质制造。 Suitable components may be glass, silicon, quartz or any other optically clear material manufactured by the substance. 对于暴露于各种分析物如核酸、蛋白质和类似物的充满流体的区室,暴露于这些分子的表面可以通过涂覆进行修饰,以使表面从疏水性转变为亲水表面,和/或降低分子在表面的吸附。 For exposure to various analytes, such as a fluid-filled compartment nucleic acids, proteins and the like, the surfaces exposed to such molecules may be modified by coating, so that the surface from a hydrophobic into a hydrophilic surface and / or lower adsorbed on the surface of the molecule. 常规芯片材料如玻璃、硅、石英和/或PDMS的表面修饰是已知的(例如美国专利US 6,263,286)。 Conventional chip materials such as glass, silicon, quartz and / or PDMS surface modification are known (e.g. U.S. Patent No. US 6,263,286). 这样的修饰包括,例如用商业可得的毛细管涂料(capillary coating)(Supelco,BellafoNTe,PA)、具有各种功能(例如聚环氧乙烷、丙烯酰胺等)的硅烷进行涂覆。 Such modifications include, for example, with commercially available capillary coatings (capillary coating) (Supelco, BellafoNTe, PA), having various functions (e.g., polyethylene oxide, acrylamide, etc.) silane coated.

[0134] 在某些实施方式中,使用这种MEMS设备制备分子条码,以从未被结合的组分中分离形成的分子条码;使分子条码暴露于靶标;和/或检测与靶标结合的分子条码。 [0134] In certain embodiments, the molecule was prepared using the bar code such MEMS devices to be incorporated in the components from the separation of molecules formed by the bar code; molecular bar code is exposed to the target; and / or detect binding to the target molecule barcode.

实施例 Example

[0135] 下面的实施例用于说明本发明的优选实施方式。 [0135] The following examples are illustrative of preferred embodiments of the present invention. 本领域的技术人员应当理解,在按照发明人所发现的本技术所进行的实施例中所公开的技术在本发明的实践中效果较好,因而可以被看作构成其实践的优选方式。 Those skilled in the art will appreciate that, in the embodiment according to the technical art of the present inventors discovered performed as disclosed in the practice of the present invention better, and thus constitute preferred modes for its practice it may be considered. 然而,本领域的技术人员根据本公开内容应当理解,在不偏离本发明的精神和范围的情况下,在公开的具体实施方式中可以进行许多改变,而仍获得类似或相似的结果。 However, those skilled in the art will appreciate from this disclosure, without departing from the spirit and scope of the present invention, in the particular embodiment disclosed that many changes may be made and still obtain a like or similar result.

实施例1.分子条码的拉曼检测 Example 1. Raman barcode detection molecule

[0136] 图3说明带有附着的拉曼标签的示例性单链条码。 [0136] FIG 3 illustrates an exemplary bar code with a single strand of a Raman label attached. 示例性寡核苷酸序列301、302、303、304通过标准的亚磷酰胺化学合成。 Exemplary oligonucleotide sequences 301, 302 standard by phosphoramidite chemical synthesis. 用于光学检测的标签被连接在寡核苷酸上,包括荧光染料ROX(羧基-X-罗丹明)310;FAM(6-羧基荧光素)320;和TAMRA(四甲基罗丹明)330。 For optical detection of the tag is attached to an oligonucleotide, comprising a fluorescent dye ROX (carboxy-rhodamine -X-) 310; FAM (6- carboxy fluorescein) 320; and TAMRA (tetramethylrhodamine) 330. 连接在每个条码上的染料标签的位置和类型如图3所示。 Location and type of each bar code attached to a dye label is shown in Fig. 在合成过程中,氨基附加在三个寡核苷酸302、303、304的5′端。 During the synthesis, an amino group attached to the three oligonucleotide 5 'ends of 302, 303.

实施例2.分子条码的拉曼光谱 Example 2. Raman spectrum of molecular barcodes

[0137] 对图3所示的分子条码进行SERS。 [0137] The bar code shown in FIG. 3 molecules for SERS. SERS发射光谱如图4所示。 SERS emission spectrum as shown in Figure 4. 样品含有220μl所示条码301、302、303、304在银凝胶和LiCl中的1μM溶液,将样品暴露于100ms的激光束,并记录表面增强拉曼光谱。 Sample solution containing 1μM barcode shown in 220μl 301,302,303,304 LiCl and silver in the gel, the samples were exposed to a laser beam of 100ms, and the recording surface enhanced Raman spectroscopy. 光谱偏移大约1000CCD计数单位。 Spectral shifting about 1000CCD counts. 如图4所示,即使三个分子条码302、303、304含有在同一寡核苷酸序列302、303、304上不同位置连接的同一拉曼标签330,但四个分子条码301、302、303、304中每一个都产生了可区别开的拉曼发射光谱。 4, even if the three barcodes 302, 303 contain the same molecular Raman tags 330 are connected at different positions on the same oligonucleotide sequence 302, 303, 301, 302, but the four molecular barcodes distinguishable, and 304 each generate Raman emission spectrum. 这证明了用本公开的方法产生可区别分子条码的可行性。 This demonstrates the feasibility of generating distinguishable molecular barcode with the disclosed methods.

实施例3 Example 3

[0138] 由附加在核苷酸上的几个示例性拉曼标签所产生的SERS光谱801、802、803、804、805、806如图8所示。 [0138] SERS attached to the nucleotide by a few exemplary Raman spectra generated 801,802,803,804,805,806 labels as shown in FIG. 各个拉曼标签所产生的光谱图801、802、803、804、805、806是容易区别的。 Raman spectra of each tag is generated 801,802,803,804,805,806 easily distinguishable. 样品含有220μl条码在银凝胶和LiCl中的1μM溶液,将样品暴露于100ms的激光束,并记录表面增强拉曼光谱。 220μl sample containing barcode 1μM solution of LiCl and silver gels, the sample is exposed to a laser beam of 100ms, and the recording surface enhanced Raman spectroscopy. 显示的是聚T[NeBu]T 801;聚T[EthdA]T 802;聚T[8Br-dA]T 803;聚T[2AmPur]T 804;[ThiSS]聚TdA 805和[5Acrd]聚dG[AmC7]806的SERS发射光谱。 Shows a poly T [NeBu] T 801; poly T [EthdA] T 802; poly T [8Br-dA] T 803; poly T [2AmPur] T 804; [ThiSS] Poly TdA 805 and [5Acrd] poly dG [ AmC7] 806 of SERS emission spectra.

实施例4 Example 4

[0139] 对本发明的一个示例性实施方式进行说明。 [0139] to an exemplary embodiment of the present invention will be described. 用译码方法确定核酸序列,如图5和图6/7所示。 Determining a nucleic acid sequence decoding method, as shown in FIG. 5 and FIG 6/7. 创建码组分文库或多个文库(图6,601、602、603、604),使得文库的每个组分都具有相伴的标记(如拉曼标签),该标记特异地、惟一地鉴定该组分(如3-mer)。 Create a code library or a plurality of component library (FIG 6,601,602,603,604), such that each component of the library are labeled with concomitant (such as Raman tags), the tag specifically, uniquely identify the component (e.g., 3-mer). 将核酸与组分库或多个文库一起温育,使探针与靶序列605杂交。 Incubating a nucleic acid library or a plurality of the component library the probe hybridizes to the target sequence 605. 杂交的核酸受控通过微流体通道,在那里,它们流过激发源和检测器。 Controlled by nucleic acid hybridization microfluidic channel, where they flow through the excitation source and detector. 检测码组分的发射光谱并传送到数据处理系统。 Emission spectrum component detection code and transmitted to the data processing system. 通过将发射光谱和发射光谱被检测的次序与结合标记的码组分的光谱数据库比较,确定核酸的序列。 Compared with the spectral database code labeled component bound by the order of the emission and emission spectra are detected, determining the sequence of nucleic acid.

[0140] 例如,可以从怀疑有疾病的对象获得组织样品(如活体解剖样品或可能的血液样品)。 [0140] For example, obtaining a tissue sample (e.g., biopsy samples or blood samples may be) from a subject suspected of having the disease. 通过本领域已知的技术可以产生单份细胞悬浮液,并用几种膜破裂缓冲液之一裂解细胞,以释放细胞内物质。 May be produced by techniques known in the art single serving cell suspension, it lysis buffer and ruptured cells with one of several membranes to release intracellular material. 用本领域已知的方法分离核酸(如苯酚/氯仿提取、凝胶纯化等)。 Isolated nucleic acids using methods known in the art (e.g., phenol / chloroform extraction, gel purification). 纯化的核酸分子通过附着在尼龙膜、96孔微量滴定板或其它固定化基质上被固定。 Purified nucleic acid molecule is fixed by adhering to the nylon membrane, 96 well microtiter plates or other immobilization matrix. 将码组分引入固定的核酸,例如一次一个或一次几个,并使其在预先确定的严紧性(NaCl含量)的缓冲液中与分子相互作用。 The fixed code component into a nucleic acid, for example one or a few time, and allowed to interact with the molecules stringency (NaCl content) of the buffer determined in advance. 将编码的探针与靶核酸杂交。 The encoding nucleic acid hybridization probes and targets. 在第一个或多个码组分杂交后,可以再加入另外的编码组分。 After the first hybridization one or more code components, can be added additional coding components. 通过大量洗涤,除去未杂交的码组分和相互杂交的码组分,只留下与固定的核酸杂交的码组分。 By extensive washing to remove unhybridized components of code symbols and each component of the hybrid, leaving only the code component of the immobilized nucleic acid hybridization. 然后逐个移去码组分并通过译码与码组分匹配的核酸序列进行阅读。 Then removed one by one by reading the code component and a nucleic acid sequence coding the code matching components. 根据期望的最终结果,测定所有或部分序列。 The end result desired, all or part of the measurement sequence. 将这些信息与被测试疾病的已知信息进行比较,具体序列的存在与否可确定相关问题疾病的对象状况。 This information is compared with the known test information is a disease, the presence or absence of the specific sequence may determine the object-related condition disease problems. 在一个例子中,与疾病相关的SNPs(单核苷酸多态性)被鉴定,从而不需要对固定的核酸进行完整测序。 In one example, the disease-associated SNPs (single nucleotide polymorphisms) were identified, so that no nucleic acid to immobilized completely sequenced.

[0141] 可选地,可将一个或多个码组分固定在表面上,如96孔平板,这些可用于捕获包含靶序列的相应核酸分子,例如已知的SNP、作为具体基因型的标志的插入或删除等。 [0141] Alternatively, the code may be one or more components immobilized on a surface, such as a 96 well plate, which can be used to capture the corresponding nucleic acid molecule comprising a target sequence, for example, the SNP is known, as a specific marker genotype insertion or deletion. 由于标签如拉曼标记的灵敏性,快速鉴定靶序列是可能的。 Since labels such as sensitivity, rapid identification of target sequences Raman labels are possible.

实施例5 Example 5

[0142] 对本发明的一个示例性实施方式进行说明。 [0142] to an exemplary embodiment of the present invention will be described. 如前所述纯化蛋白质或肽(例如稀有的调节蛋白质等)。 Purified protein or peptide as described above (e.g., regulatory proteins like rare). 然后,使用本领域熟知的技术,用纯化的蛋白质/肽产生抗体(单克隆抗体也可用本领域已知的技术产生)(Antibodier:ALaboratory Manual,Cold Spring Harbor Laboratory,Cold Spring Harbor Press,ColdSpring Harbor,NY,1988)。 Then, using techniques known in the art, with purified protein / peptide to produce antibodies (monoclonal antibodies may also be generated techniques known in the art) (Antibodier: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, ColdSpring Harbor, NY, 1988). 通过一同引入佐剂如弗氏完全或不完全佐剂,可提高抗原的反应性。 By introducing such as Freund's complete adjuvant or with incomplete adjuvant, can increase the reactivity of the antigen. 将抗原附着于载体,例如牛血清清蛋白、匙孔血蓝蛋白,可提高抗原性。 The antigen attached to a carrier, such as bovine serum albumin, keyhole limpet hemocyanin, improved antigenicity. 通过周期性地强化注射抗原,可提高动物的免疫响应。 By periodic booster injections of antigen, can enhance the immune response of animals. 抗体1206分泌进入动物的循环系统,并通过出血或心脏穿刺(cardiac puncture)获取。 1206 Antibodies secreted into the circulatory system of an animal, and obtained by bleeding or cardiac puncture (cardiac puncture). 抗体1206用熟知的方法与其它血液组分分离,例如血液凝块、离心、过滤和/或免疫亲和纯化(如采用抗兔抗体)或亲和层析(例如,蛋白A琼脂糖柱层析)。 1206 antibody well-known methods of separating the other blood components, such as blood clots, centrifugation, filtration and / or immunoaffinity purification (e.g., using anti-rabbit antibodies) or affinity chromatography (e.g., Protein A Sepharose column chromatography ). 然后将这些抗体与图12所示的任何一个聚合物拉曼标记连接(例如,共价地连接)。 Raman any one polymer are then shown in FIG. 12 labeled with these antibodies is connected (e.g., covalently linked). 然后用聚合物拉曼标记抗体鉴定具有许多分子的提取物中的蛋白质。 Then the extract having many molecules the protein with a labeled antibody identification Raman polymer. 可选地,可用聚合物拉曼标记抗体从众多分子的提取物中分离出几个相同的蛋白质,目的在于鉴定、进一步研究感兴趣的蛋白质;阻遏蛋白质的活性;鉴定与疾病相关的蛋白质等。 Alternatively, the polymer can be used Raman label antibodies isolated from the extract and the molecules of the several of the same protein, the aim to identify further study of the protein of interest; repressor activity of the protein; such as protein identification associated with the disease. 由于聚合物拉曼标记分子(如聚合物拉曼标记Ab)容易被检测,因此也可将它用于诊断目的,以评估疾病的存在和/或程度。 Since the polymer Raman label molecules (e.g., polymeric Raman label Ab) is easily detected, and therefore it can also be used for diagnostic purposes to assess the presence of disease and / or extent.

实施例6.用图5-7所示的技术鉴定核酸序列: Example 6. Identification of a nucleic acid sequence shown in FIG. 5-7 art:

[0143] 在一个实施方式中,用一个或多个拉曼标签修饰核酸。 [0143] In one embodiment, the one or more Raman labels with modified nucleic acid. 许多小而独特的拉曼标签是有效的。 Many small and unique Raman tags are valid. 在一个例子中,图13说明了具有强而独特的拉曼标记的几个腺嘌呤类似物(其它如图8所示)。 In one example, FIG. 13 illustrates the strong and unique Raman label several adenine analogues (other as shown in FIG. 8). 在一个例子中,将拉曼标签通过一个或多个碱基修饰与核酸连接,然后用这些修饰的碱基制造亚磷酰胺,用于寡核苷酸的化学分析。 In one example, the one or more Raman tags linked to nucleic acid base modification, then these modified bases for producing phosphoramidite, oligonucleotides used for chemical analysis. 修饰碱基的亚磷酰胺可用本领域已知的技术制造(McBride,LJ和Caruthers,MH(1983)“An investigation of several deoxynucleosidephosphoramidites useful for synthesizing deoxyoligonucleotides.”Tetrahedron Lett.24:245-248)。 Modified base phosphoramidites available manufacturing techniques known in the art (McBride, LJ, and Caruthers, MH (1983) "An investigation of several deoxynucleosidephosphoramidites useful for synthesizing deoxyoligonucleotides." Tetrahedron Lett.24: 245-248).

[0144] 在一个实施方式中,码组分可以由大约10-20个碱基的长度组成。 [0144] In one embodiment, the component code may consist of a length of about 10-20 bases. 对于10-mer,这有4^10个可能的序列。 For the 10-mer, which has 4 ^ 10 possible sequences. 对于20-mer,这有4^20个可能的序列。 For the 20-mer, which has 4 ^ 20 possible sequences. 在实际应用中,靶序列(多个靶序列)是已知的或者序列可以分成序列子集。 In practical applications, the target sequence (s target sequence) are known or may be divided into a sequence a subset of sequences. 因此,例如,可用一个或多个拉曼标签标记和鉴别寡核苷酸。 Thus, for example, using one or more Raman labels and identification marks oligonucleotides. 在一个例子中,如果使用10个不同的亚磷酰胺(每个具有不同的拉曼标签),则如果每个合成的寡核苷酸序列有1个拉曼标签,那么可合成10个不同的寡聚体(oligo);如果每个合成的寡核苷酸有2个拉曼标签,那么可合成55个寡聚体;如果每个寡核苷酸有3个标签,那么可合成175个寡聚体。 In one example, if using 10 different phosphoramidite (each having a different Raman tag), then if each synthetic oligonucleotide sequence has a Raman label, it can be synthesized ten different oligomer (oligo); if each synthetic oligonucleotide has 2 Raman labels, it can be synthesized oligomer 55; if there are three each oligonucleotide tag, the oligonucleotides can be synthesized 175 mer. 例如,可使用亚磷酰胺合成寡核苷酸(码组分),这些方法是本领域知晓的。 For example, a phosphoramidite synthesis of oligonucleotides (component codes), these methods are known in the art. 举一个例子,一个组分可为ATGCGACGT(SEQ ID NO:3),以呋喃甲基氨基嘌呤(KN)为标签(图13),而另一个为GCTATAGCCG(SEQ IDNO:4),以苯甲酰基-腺嘌呤(BA)为标签(图13)。 In one example, a component may be ATGCGACGT (SEQ ID NO: 3), to furylmethyl diaminopurine (KN) of the label (FIG. 13), and the other is GCTATAGCCG (SEQ IDNO: 4), benzoyl group - adenine (BA) as the label (FIG. 13). 许多条码组分可预先制备并存放备用。 Many bar code components may be prepared in advance and stored for use.

[0145] 在一个实施方式中,用下面的方法制备条码。 [0145] In one embodiment, the bar code was prepared by the following method. 条码可以由几个码组分组装而成。 Barcode code may be assembled from several components. 条码模板可以是相对较长的多核苷酸,例如40个核苷酸的DNA片段,其可以用标准的亚磷酰胺化学方法合成:5′ACGTCGCATT-CGGCTATAGC-TTTCTATAGCGCTATGGTAC 3′(SEQ ID NO:5) Barcode template may be relatively long polynucleotides such as a DNA fragment of 40 nucleotides, which can be used phosphoramidite chemistry standard synthetic: 5'ACGTCGCATT-CGGCTATAGC-TTTCTATAGCGCTATGGTAC 3 '(SEQ ID NO: 5)

[0146] 本例中下划线部分是容器部分,其它序列是探针部分。 [0146] In the present embodiment the underlined portion is a container portion, the other part of the probe sequence. 条码组分5′-ATGCGACGT(KN)-3′(SEQ ID NO:3)和5′-GCTATAGCCG(BA)-3′(SEQ IDNO:4)在标准条件下与容器部分杂交(例如,在200mM NaCl、10mM TrisHCl、pH7.5和1mM EDTA存在下,寡核苷酸的浓度为1到10μM)。 Barcode component 5'-ATGCGACGT (KN) -3 '(SEQ ID NO: 3) and 5'-GCTATAGCCG (BA) -3' (SEQ IDNO: 4) under standard hybridization conditions to the container portion (e.g., at 200mM under NaCl, 10mM TrisHCl, pH7.5 and 1mM EDTA present, oligonucleotide concentration of 1 to 10μM). 因此,在本例中,探针部分由2条码组分表示,并通过将呋喃甲基氨基嘌呤和苯甲酰基-腺嘌呤都作为拉曼标签,测定其拉曼标记。 Accordingly, in the present embodiment, the probe component parts are denoted by two yards, and by amino-purin-furylmethyl and benzoyl - adenine as Raman labels are measured for Raman label. 为合成不同的条码模板,探针部分和容器部分相应地改变;不同的条码组分(预先制备)杂交在一起,形成新的条码。 Templates for the synthesis of different barcodes, the probe portion and the container portion correspondingly change; different barcode components (previously prepared) hybridized together to form a new barcode.

[0105] 该技术可以用于,例如,通过分析对应于传染性物质的DNA或RNA的存在,检测传染性物质。 [0105] This technique can be used, for example, by analyzing the DNA or RNA corresponding to the presence of infectious material, detecting infectious agents. 在收集到样品并通过本领域已知技术从样品中提取核酸之后,将核酸消化(例如,通过限制酶、有限DNA酶消化等),在生物素化ddNTP(Perkin Elmer Life Sciences)存在时,通过Terminal Transferase(来自New EnglandBiolabs)用生物素进行末端标记。 After the sample is collected and the nucleic acid extracted from the sample by known techniques in the art, the nucleic acid digestion (e.g., by restriction enzymes, DNA is limited enzyme digestion, etc.), in the presence of biotinylated ddNTP (Perkin Elmer Life Sciences), by terminal Transferase (from New EnglandBiolabs) was end-labeled with biotin. 在用凝胶过滤柱(Amersham-Pharmacia Biotech)去除自由核苷酸后,生物素化的DNAs被捕获在包被有链霉抗生物素的磁性珠子(Roche)上。 After removal of the free nucleotides by gel filtration column (Amersham-Pharmacia Biotech), biotinylated DNAs were captured on streptavidin-coated with avidin magnetic beads (Roche). 然后用0.1N NaOH(对DNA)使核酸变性,分离2个互补链。 Then treated with 0.1N NaOH (for DNA) nucleic acid denaturation, two separated complementary strands. 在中和靶分子后,引入条码分子,以便结合互补序列。 After the target molecule is introduced barcode molecule, binding to the complementary sequence. 结合/洗涤缓冲液的一个例子是200mM NaCl、10mM TrisHCl、pH7.5和1mM EDTA。 Examples of a binding / wash buffer is 200mM NaCl, 10mM TrisHCl, pH7.5, and 1mM EDTA. 使用本领域已知的方法,可使用磁体(Dynal Corp)控制颗粒。 Using methods known in the art, using a magnet (Dynal Corp) control particles.

[0106] 在一个例子中,条码的探针部分与靶序列互补,例如,5'GTACCATAGCGCTATAGAAA 3'(SEQ ID NO:6)条码分子将与靶序列结合,从而通过本例中的磁体(Dyna珠子,Dyna)而被保留。 [0106] In one example, the target probe portion is complementary to the barcode sequences, e.g., 5'GTACCATAGCGCTATAGAAA 3 '(SEQ ID NO: 6) molecules will bind to the target bar code sequence, so that by this embodiment a magnet (Dyna beads , Dyna) are retained. 将珠子与银胶体(由1mMAgNO3,用水1∶2稀释制得)和0.1M LiCl(最终浓度)混合。 The beads and silver colloid (final concentration) (manufactured by 1mMAgNO3, 1:2 prepared was diluted with water) and 0.1M LiCl. 当颗粒通过拉曼检测器时,就可以检测与条码分子特定地结合的拉曼信号(KN和BA)。 When the particles by a Raman detector, Raman signal can be detected specifically bound to the molecules of the bar code (KN and BA). 在本例中,该信息被用于确认一个或多个样品中具体传染性物质存在与否。 In the present embodiment, the specific information is used to confirm the presence or absence of one or more infectious agents in a sample.

实施例7用于检测蛋白质的条码-抗体 Example 7 yards for detecting protein - antibody

[0107] 另一个实施方式包括,如实施例6制备拉曼标记条码(或多个条码),而条码然后与抗体连接,用于检测抗原(如蛋白质)。 [0107] Another embodiment includes, as in Example 6 Preparation of Raman label barcode (barcode or more), and the bar code and then linked to the antibody, for the detection of antigens (e.g., proteins). 由此,制备条码制剂并制造DNA标记的抗体。 Thus, a bar code and producing the antibody preparation labeled DNA. 例如,用在200μl 0.1xPBS(稀释自10xPBS,从Ambion获得)中的20μg(52纳摩尔)磺基-GMBS(Pierce Cat.No.22324)在37℃时活化IgG抗体(例如,200μg(1.33纳摩尔))30min,再在室温下活化30min。 For example, in 200μl 0.1xPBS (diluted from 10xPBS, obtained from Ambion) IgG antibody 20μg (52 nmoles) of sulfo -GMBS (Pierce Cat.No.22324) activated at 37 [deg.] C (e.g., 200μg (1.33 nano mol)) 30min, 30min at room temperature reactivation. 然后使溶液通过PD-10柱(Amersham-Pharmacia),收集含有抗体的组分。 Then passing the solution through PD-10 column (Amersham-Pharmacia), antibody-containing fractions were collected. 硫醇修饰的DNA寡聚物由商家合成(Qiagen-Operon)。 Thiol-modified DNA oligomers were synthesized by the merchant (Qiagen-Operon). 在根据商家的说明还原二硫键(如DTT处理)后,将DNA寡聚物(如13纳摩尔)与纯化且活化的抗体混合。 The merchant's instructions after reduction of disulfide bonds (e.g., treatment with DTT), the DNA oligomer (e.g., 13 nmol) was mixed with purified antibodies and activation. 反应在室温进行2小时并在4℃过夜。 The reaction for 2 hours at room temperature and overnight at 4 ℃. 然后用离子交换柱(Amersham-Pharmacia)纯化DNA-抗体,使用例如0-2M NaCl梯度。 It was then purified by ion exchange DNA- antibody, e.g. 0-2M NaCl gradient column (Amersham-Pharmacia). 收集既含有DNA又含有蛋白质的组分。 Containing both DNA and collecting fractions containing protein. 在采用本领域已知的技术进行脱盐和浓缩处理后,将样品用于抗原的结合(蛋白质检测)。 After using techniques known in the art desalting and concentration treatment, the sample for binding to an antigen (protein detection).

[0108] 几种方法可用于将抗原(如蛋白质)固定在固体支持物上。 [0108] Several methods are available for antigens (e.g., protein) immobilized on a solid support. 优选地,对于拉曼检测,可通过EDC化学方法,将捕获的抗体固定在金表面上(捕获抗体和检测抗体应当识别同一抗原,其可从商家获得,例如R&D System和BDBiosciences)(Benson等,Science,193,(2001),1641-1644)。 Preferably, for Raman detection, the antibody will capture fixed by EDC chemical method on the gold surface (the capture antibody and a detection antibody should recognize the same antigen, which is available from a merchant, e.g. R & amp; D System and BDBiosciences) (Benson etc., Science, 193, (2001), 1641-1644). 将含有靶抗原(如蛋白质)的样品稀释在1xPBS中,然后施用于固体表面,进行特异性结合。 Sample containing the target antigen (e.g., protein) diluted in 1xPBS, and then applied to the solid surface, specific binding. 例如,使DNA标记抗体与捕获的抗原(如蛋白质靶标)结合。 For example, the DNA labeled antibody to the captured antigen (e.g., protein targets) binding. 然后进行标准的免疫测试过程,一般地,使用1xPBS和0.05%Tween-20(吐温-20)。 Then standard immunoassay process, in general, the use of 1xPBS and 0.05% Tween-20 (Tween-20). 一旦发生了结合,就可将互补的拉曼标记DNA与固定的与检测抗体相连的DNA寡聚物相结合。 Once binding occurs, complementary Raman label can be combined with DNA immobilized DNA oligomer coupled with the detection antibody. 一般地,条码分子在2xPBS和1□g/ml酵母tRNA(Sigma)中的浓度可以为10nM。 Generally, the concentration of molecules in the barcode and 2xPBS 1 □ g / ml yeast tRNA (Sigma) may be as 10nM. 在用1xPBS洗涤之后,将银胶体(由1mM AgNO3,用水1∶2稀释制得)加入结合表面,然后将LiCl加到0.1M,接着进行拉曼测定。 After washing with 1xPBS, silver colloid (a 1mM AgNO3, diluted 1 prepared with water) was added bonding surface, and the LiCl was added to 0.1M, followed by a Raman measurement. 由于与抗体连接的DNA寡聚物与条码的探针部分互补,因此条码标记的存在将表明抗体的存在,进而表明靶抗原(蛋白质)的存在。 Since DNA oligomer probe portion and the bar code attached to the antibody is complementary, there is code marking will indicate the presence of antibodies, thus indicating the presence of the target antigen (protein). 当不同的捕获抗体和DNA标记检测抗体用于同一系统中时,用这种方法可同时检测几个不同的抗原。 When different capture antibodies and detection antibodies labeled DNA used in the same system, this method can simultaneously detect several different antigens.

[0109] 根据本公开内容,在没有不适当的实验方法的情况下,可制造和使用这里公开的和要求保护的所有方法、组合物和设备。 [0109] According to the present disclosure, without undue experimentation case, and may be manufactured using all the methods herein disclosed and claimed, the composition and equipment. 对本领域技术人员来说,显而易见的是,在不偏离要求保护主题的概念、精神和范围的情况下,可以对这里所述的方法、组合物和设备进行改变。 The skilled person, will be apparent that, in the case of the concept, without departing from the spirit and scope of the claimed subject matter, changes may be made to the methods described herein, compositions and devices. 更具体地,显然,化学及生理学关联的某种试剂(物质)可以替代这里所述的试剂(物质),同时获得相同的或相似的结果。 More specifically, it is clear, chemically and physiologically associated with an agent (substance) may be substituted for the agents (materials) described herein while the same or similar results. 对本领域技术人员显而易见的所有这些类似的替代和修饰都被认为在所要求保护主题的精神、范围和概念之内。 Obvious to those skilled in the All such similar substitutes and modifications are to be considered within the spirit of the claimed subject matter, scope and concept.

Claims (34)

1.一种方法,包括:获得条码,所述条码包括与有机分子骨架相连的两个或更多个标签;将所述条码与靶结合;以及检测与所述靶结合的所述条码。 1. A method, comprising: obtaining bar code, the bar code label comprises two or more organic molecules attached to the backbone; the barcode bind to the target; and detecting the binding of the target bar code.
2.如权利要求1所述的方法,其中所述骨架包括选自核酸、肽、多糖、生物聚合物和合成聚合物中的至少一种分子。 2. The method according to claim 1, wherein said backbone comprises at least one molecule selected from nucleic acids, peptides, polysaccharides, biopolymers, and synthetic polymers.
3.如权利要求2所述的方法,其中所述核酸是单链DNA。 3. The method according to claim 2, wherein said nucleic acid is single stranded DNA.
4.如权利要求2所述的方法,其中所述骨架包括共价连接于肽的核酸。 4. The method according to claim 2, wherein said backbone comprises a nucleic acid covalently attached to a peptide.
5.如权利要求1所述的方法,其中所述标签选自核酸、核苷酸、核苷酸类似物、碱基类似物、荧光染料、肽、氨基酸、修饰氨基酸、有机部分、拉曼标签、量子点、碳纳米管、富勒烯、亚微米金属颗粒、电子致密颗粒和晶体颗粒。 5. The method according to claim 1, wherein said label is selected from nucleic acids, nucleotides, nucleotide analogs, base analogs, fluorescent dyes, peptides, amino acids, modified amino acids, organic moieties, Raman label , quantum dots, carbon nanotubes, fullerenes, submicrometer metallic particles, electron dense particles and crystalline particles.
6.如权利要求1所述的方法,其中所述骨架包括一种或多种支化的核酸。 6. The method according to claim 1, wherein said backbone comprises one or more branched nucleic acids.
7.如权利要求6所述的方法,其中支链位于骨架上预先确定的位置。 7. The method according to claim 6, wherein the branched backbone located predetermined positions.
8.如权利要求1所述的方法,其中所述条码用选自荧光光谱法、拉曼光谱法、傅里叶变换红外光谱法(FTIR)和表面等离子体共振法的技术进行检测。 8. The method according to claim 1, wherein said bar code is selected by fluorescence spectroscopy, Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR) techniques and surface plasmon resonance is detected.
9.如权利要求1所述的方法,其中所述条码通过探针部分与靶结合。 9. The method according to claim 1, wherein said bar code through the probe binding to the target portion.
10.如权利要求1所述的方法,其中可区分的条码通过同一标签连接在同一骨架上不同位置而产生。 10. The method according to claim 1, produced at different positions on the same backbone wherein distinguishable bar code label through the same connection.
11.如权利要求1所述的方法,其中所述靶选自蛋白质、肽、糖蛋白、脂蛋白、感染性蛋白、核酸、多核苷酸、寡核苷酸、脂、脂肪酸、糖类、糖脂、磷脂、鞘脂、脂多糖、多糖、真核细胞、原核细胞、细菌、噬菌体、病毒和病原体。 11. The method according to claim 1, wherein said target is selected from proteins, peptides, glycoproteins, lipoproteins, infectious proteins, nucleic acids, polynucleotides, oligonucleotides, lipids, fatty acids, saccharides, sugar fat, phospholipids, sphingolipids, lipopolysaccharides, polysaccharides, eukaryotic cells, prokaryotic cells, bacteria, bacteriophages, viruses, and pathogens.
12.一种方法,包括:获得核酸模板,所述核酸模板包括容器部分和探针部分;和将一种或多种标记寡核苷酸与所述容器部分杂交,形成条码。 12. A method, comprising: obtaining a nucleic acid template, the nucleic acid template includes a container portion and a probe portion; and one or more labeled oligonucleotide hybridize to a portion of the container, the bar code is formed.
13.如权利要求12所述的方法,进一步包括将所述条码与靶结合。 13. The method of claim 12, further comprising the bar code to the target binding.
14.如权利要求13所述的方法,进一步包括检测与所述靶结合的所述条码。 14. The method according to claim 13, further comprising detecting a bar code of the target binding.
15.制造聚合物拉曼标记的方法,包括:获得两个或多个单体单元;和聚合所述单体单元,形成聚合物拉曼标记。 15. A method for producing a polymer Raman labels, comprising: obtaining two or more monomer units; and polymerizing the monomer unit forming the polymer Raman label.
16.如权利要求15所述的方法,其中每一单体单元包括拉曼标签。 16. The method according to claim 15, wherein each monomer unit comprises a Raman label.
17.如权利要求16所述的方法,其中在单个聚合物拉曼标记中的每个拉曼标签是不同的。 17. The method according to claim 16, wherein each tag in a single polymer Raman Raman labels are different.
18.如权利要求16所述的方法,其中所述拉曼标签通过间隔部分与聚合物拉曼标记的骨架连接。 18. The method according to claim 16, wherein the Raman tag is attached through a spacer moiety backbone polymer Raman labels.
19.如权利要求15所述的方法,其中在单体单元的聚合之后,将拉曼标签与聚合物拉曼标记连接。 19. The method according to claim 15, wherein the monomer unit after polymerization, the polymeric Raman label Raman labels connection.
20.如权利要求15所述的方法,进一步包括,在单体单元的一端去保护功能基,以及在所述单体单元与所述聚合物拉曼标记之间形成共价键。 20. The method according to claim 15, further comprising, at an end of the monomer units to protect functional groups, and forming covalent bonds between the monomer units in the polymer Raman labels.
21.如权利要求15所述的方法,进一步包括产生次级聚合单元,每个次级聚合单元包括预先确定数目的单体单元。 21. The method according to claim 15, further comprising generating a secondary polymerized units, each secondary unit comprising polymerized monomer units of predetermined number.
22.如权利要求15所述的方法,进一步包括将所述聚合物拉曼标记与固体支持物连接。 22. The method according to claim 15, further comprising the polymeric Raman tag connected to the solid support.
23.如权利要求22所述的方法,其中所述固体支持物是纳米颗粒或珠子。 23. The method according to claim 22, wherein said solid support is nanoparticles or beads.
24.如权利要求15所述的方法,进一步包括将探针连接在所述聚合物拉曼标记上。 24. The method according to claim 15, further comprising a probe attached to the polymeric Raman label.
25.如权利要求24所述的方法,进一步包括将所述探针与靶结合。 25. The method according to claim 24, further comprising the probe bound to a target.
26.如权利要求25所述的方法,进一步包括检测与所述靶结合的所述探针。 26. The method according to claim 25, further comprising detecting said probe bound to said target.
27.一种聚合物拉曼标记,其包括:两个或更多个单体单元,其相互共价地连接;两个或更多个拉曼标签;和至少一个探针。 27. A polymer Raman labels, comprising: two or more monomeric units, which are covalently linked; two or more Raman labels; and at least one probe.
28.如权利要求27所述的聚合物拉曼标记,进一步包括与所述聚合物拉曼标记连接的纳米颗粒或珠子。 Polymeric Raman label of claim 27, further comprising nanoparticles or beads labeled with the connector 28. The polymer Raman claims.
29.如权利要求27所述的聚合物拉曼标记,其中标记中每个拉曼标签是不同的。 29. The polymeric Raman label according to claim 27, wherein each tag is a different Raman tag.
30.如权利要求27所述的聚合物拉曼标记,进一步包括每个拉曼标签的两个或更多个拷贝。 Polymeric Raman label of claim 27, further comprising each of two or more Raman labels as claimed in claim 30. copies.
31.一种系统,其包括:成像仪器;至少一个与探针连接的条码;和至少一个与所述探针结合的靶。 31. A system, comprising: an imaging apparatus; at least a bar code attached to the probe; and at least one target binding to the probe.
32.如权利要求31所述的系统,其中所述成像仪器选自荧光仪器、拉曼仪器和FTIR仪器。 32. The system according to claim 31, wherein said imaging apparatus is selected from fluorescence instrument, Raman and FTIR instrument instrument.
33.如权利要求31所述的系统,其中每个条码包括两个或更多个拉曼标签。 33. The system according to claim 31, wherein each barcode comprises two or more Raman labels.
34.如权利要求33所述的系统,其中在单个条码上的每个拉曼标签具有不同的拉曼发射光谱。 34. The system according to claim 33, wherein each Raman labels on a single bar code with a different Raman emission spectra.
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